G protein coupled receptor agonists and antagonists and methods of activating and inhibiting g protein coupled receptors using the same

ABSTRACT

The invention relates generally to G protein coupled receptors and in particular to agonists and antagonists of G protein receptors and methods of using the same.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/606,368, filed on Nov. 28, 2006, which is a continuation of U.S.patent application Ser. No. 10/251,703, filed on Sep. 20, 2002, which isa continuation-in-part of U.S. patent application Ser. No. 09/841,091,filed on Apr. 23, 2001, now U.S. Pat. No. 6,864,229, which claims thebenefit of U.S. Provisional Patent Application No. 60/198,993, filed onApr. 21, 2000, each of which is incorporated by reference herein in itsentirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. Government support under NationalInstitutes of Health grants R01HL64701 and R01HL57905. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to G protein coupled receptors and inparticular to agonists and antagonists of G protein receptors andmethods of using the same.

BACKGROUND OF THE INVENTION

A variety of hormones, neurotransmitters and biologically activesubstances control, regulate, or adjust the functions of living bodiesvia specific receptors located in cell membranes. Many of thesereceptors mediate the transmission of intracellular signals byactivating guanine nucleotide-binding proteins (G proteins) to which thereceptor is coupled. Such receptors are generically referred to as Gprotein coupled receptors (“GPCR”s). Binding of a specific signalingmolecule to the GPCR can cause a conformational change in the receptor,resulting in a form that is able to bind and activate a G protein,thereby triggering a cascade of intracellular events that eventuallyleads to a biological response. Typically, GPCRs interact with Gproteins to regulate the synthesis of intracellular second messengerssuch as cyclic AMP, inositol phosphates, diacylglycerol and calciumions.

GPCRs play a vital role in the signaling processes that control cellularmetabolism, cell growth and motility, adhesion, inflammation, neuronalsignaling, and blood coagulation. G protein coupled receptor proteinsalso have a very important role as targets for a variety of signalingmolecules which control, regulate, or adjust the functions of livingbodies.

Known GPCR agonists and antagonists act on the extracellular surface ofthe GPCR. However, there are currently no effective strategies todirectly study the mechanism of receptor-G protein coupling in acontrolled fashion under in vivo conditions. Nor is there anunderstanding of the selective contacts between receptors and Gproteins, or the elucidation of the mechanisms of G protein activationby receptors.

A need remains in the art for compositions which are useful to modulateGPCR activity, and also to elucidate and further define a generalstrategy for development and screening of novel therapeutics targeted toG-protein coupled receptor-effector interfaces.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that attachment of acell-penetrating or cell membrane-associating moiety to peptides derivedfrom a GPCR produces agonists and/or antagonists of receptor-G proteinsignaling. These modified peptides—termed pepducins—exhibit selectivityfor their cognate receptor. Pepducins for protease-activated receptors(PARs), e.g., PAR1, PAR2, and PAR4, cholecystokinins A and B (CCKA,CCKB), somatostatin-2 (SSTR2), melanocortin-4 (MC4R), glucagon-likepeptide-1 receptor (GLP-1R), and P2Y₁₂ ADP receptor are agonists and/orantagonists for the receptors from which they are derived. Thesecompositions are useful to activate or inhibit the activity of a broadrange of GPCRs. Human PARs include PAR1 (Genbank Accession NumberAF019616); PAR2 (Genbank Accession Number XM_(—)003671); PAR3 (GenbankAccession Number NM_(—)004101); and PAR4 (Genbank Accession NumberNM_(—)003950.1), the sequences of which are hereby incorporated byreference.

Accordingly, the invention provides a composition containing apolypeptide component which includes an isolated fragment of a G-proteincoupled receptor (GPCR) linked to a cell penetrating component or acomponent that associates with a cell surface membrane (a membranetethering moiety).

A naturally-occurring GPCR is a cell surface molecule that crosses acell membrane at least once. For example, many naturally-occurring GPCRscross a cell membrane seven times and contain several intracellulardomains. Preferably, the isolated fragment of a GPCR includes anintracellular domain of a GPCR, e.g., one of more of the followingdomains: a first intracellular loop, a second intracellular loop, athird intracellular loop, and a C-terminal cytoplasmic domain. Theintracellular portion is selected from an intracellular domain of aone-transmembrane domain G-protein coupled receptor of a cytokine GPCR,or a fragment thereof, or an intracellular domain of amulti-polypeptide-GPCR, such as a GPIb/V/IX receptor or a collagenreceptor.

Alternatively, the isolated GPCR fragment includes an extracellularportion of a GPCR which can include a portion of the N-terminalextracellular domain, the second extracellular loop, the thirdextracellular loop, or the fourth extracellular loop. In otherembodiments, the GPCR fragment includes the cytoplasmic tail of the GPCRor a portion of the ligand binding site of the GPCR.

Thus, the invention provides a composition containing a polypeptidewhich includes an isolated fragment of a G-protein coupled receptor(GPCR) linked to a non-peptidic cell penetrating moiety. Alternatively,the invention provides a composition containing a polypeptide whichincludes an isolated fragment of a G-protein coupled receptor (GPCR)linked to a non-peptidic membrane-tethering moiety.

The invention also provides a composition containing a polypeptide whichincludes an isolated intracellular fragment of a G-protein coupledreceptor (GPCR) or an isolated extracellular fragment of a G-proteincoupled receptor (GPCR) linked to a cell penetrating moiety.Additionally, a composition which includes polypeptide which is anisolated intracellular fragment of a G-protein coupled receptor (GPCR)or an isolated extracellular fragment of a G-protein coupled receptor(GPCR) linked to a membrane-tethering moiety.

The invention also includes soluble chimeric polypeptides. Thesepeptides include a first domain which includes an extracellular or anintracellular fragment of a GPCR, and a second domain, linked to thefirst domain, which includes a cell-penetrating or a membrane-tetheringmoiety. The cell-penetrating- or membrane-tethering moiety optionallyincludes a naturally occurring contiguous amino acid from atransmembrane domain adjacent to the extracellular or intracellularfragment. For example, the construct contains at least 3, but less than16 contiguous amino acids of a GPCR transmembrane helix domain. Thetransmembrane domain is not transmembrane domain 1-7 of the CXCR4,transmembrane domain 1-7 of CCKA receptor, or transmembrane 2 of theCCR5 receptor.

In one embodiment, the soluble chimeric polypeptide has a first domainwhich includes an extracellular or an intracellular fragment of a GPCR,and a second domain containing cell-penetrating or a membrane-tetheringmoiety, linked to the first domain. The second domain includes 1-15contiguous amino acids of a naturally-occurring transmembrane helixdomain immediately adjacent to the extracellular or intracellularfragment.

A GPCR is a membrane protein which binds to a signaling molecule. Uponbinding, a conformational change occurs, which allows binding of theGPCR to, and activation of, a G-protein. The activated G-protein theninteracts with an effector molecule, which is typically involved in asecond messenger pathway. A GPCR agonist is a composition that activatesa GPCR to mimic the action of the endogenous signaling molecule specificto that receptor. A GPCR antagonist is a composition that inhibits GPCRactivity. GPCR activity is measured by ability to bind to an effectorsignaling molecule such as G-protein. An activated GPCR is one, which iscapable of interacting with, and activating a G-protein. An inhibitedreceptor has a reduced ability to bind extracellular ligand and/orproductively interact with, and activate a G-protein.

The present invention also provides an inhibitor of platelet activation,comprising a polypeptide comprising an isolated platelet-inhibitoryfragment of a protease activated receptor and a cell penetrating moietylinked to said polypeptide.

In one embodiment, the inhibitor of platelet activation comprises anisolated platelet-inhibitory fragment of a protease activated receptor,wherein the protease activated receptor is a thrombin receptor, atrypsin receptor, a clotting factor Xa receptor, an activated protein Creceptor, a tryptase receptor, or a plasmin receptor. In anotherembodiment, the thrombin receptor is PAR-4 or PAR-1. In yet anotherembodiment, the said polypeptide is SEQ ID NO: 29 or SEQ ID NO: 4. Inyet another embodiment, the inhibitor of platelet activation furthercomprises P4pal10. In yet another embodiment, the inhibitor of plateletactivation further comprises P1pal12.

The present invention also provides methods of inhibiting plateletaggregation, comprising contacting a platelet with a compositioncomprising an isolated fragment of a protease activated receptor linkedto a cell penetrating moiety. In one embodiment, the protease activatedreceptor is a thrombin receptor. Optionally, the thrombin receptor is aPAR-1 receptor or a PAR-4 receptor.

The present invention also provides methods of inhibiting thrombusformation in a mammal, comprising administering to said mammal acomposition comprising an isolated fragment of a thrombin receptorlinked to a cell penetrating moiety. In one embodiment, the isolatedfragment of a thrombin receptor is a PAR-4 or a PAR-1 receptor. In oneembodiment, said composition is infused into a vascular lumen. Forexample, the lumen is in a jugular vein or a peripheral vein. In yetanother embodiment, said composition is infused into a perivascularspace. In another embodiment, said composition is administeredtransdermally, subdermally, or subcutaneously. In one example, saidcomposition is administered to a lung tissue of said mammal. In anotherembodiment, said composition is administered into the peritoneal cavityof said mammal. In yet another embodiment, said composition isadministered vaginally or rectally to said mammal.

The present invention also provides a vascular endoprosthetic device,comprising an inhibitor of thrombus formation, said inhibitor comprisingan isolated fragment of a thrombin receptor linked to a cell penetratingmoiety. In one embodiment, said device is a stent. In anotherembodiment, said device is a catheter.

In another embodiment, said device is impregnated with said inhibitor.In yet another embodiment, said device is coated with said inhibitor.

The present invention also provides a method of inhibiting migration orinvasion of a tumor cell, comprising contacting said tumor cell with anisolated fragment of a protease activated receptor linked to a cellpenetrating moiety. In one embodiment, the isolated fragment of aprotease activated receptor is a PAR-4, PAR-2 or a PAR-1 receptor. Inanother embodiment, the isolated fragment of a protease activatedreceptor comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8,or SEQ ID NO:29. In another embodiment, method of inhibiting migrationor invasion of a tumor cell, comprises contacting said tumor cell withan isolated fragment of a protease activated receptor linked to a cellpenetrating moiety, which comprises P4pal10, P1pal12, P2pal21, P2pal21F,or P1pal7.

The present invention also provides a method of inhibiting metastases ofa tumor cell, comprising contacting said tumor cell with an isolatedfragment of a protease activated receptor linked to a cell-penetratingmoiety. In one embodiment, the isolated fragment of a protease activatedreceptor is a PAR-4, PAR 2, or a PAR-1 receptor. In another embodiment,the isolated fragment of a protease activated receptor comprises SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:29. In yetanother embodiment, the instant method of inhibiting metastases of atumor cell, comprises contacting said tumor cell with an isolatedfragment of a protease activated receptor linked to a cell-penetratingmoiety which comprises P4pal10, P1pal12, P2pal21, P2pal21F, or P1pal7.In yet another embodiment, the instant method of inhibiting metastasesof a tumor cell applies to tumor cell selected from the group consistingof a melanoma cell, a lung cancer cell, a breast cancer cell, a coloncancer cell, a central nervous system cancer cell, a liver cancer cell,a stomach cancer cell, a renal cancer cell, a prostate cancer cell, asarcoma cell, a leukemia cell, or a lymphoma cell.

The present invention also provides a method of inhibiting asthma in asubject, comprising administering to said subject a compositioncomprising an isolated fragment of a thrombin or trypsin/tryptase GPCRlinked to a cell penetrating moiety. In one embodiment, the isolatedfragment of a GPCR is a PAR-1, PAR-2 or PAR-4 receptor. In anotherembodiment, the isolated fragment of a GPCR comprises SEQ ID NO:3, SEQID NO:4, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:29. In yet anotherembodiment, said composition comprises P4pal10, P1pal12, P2pal21,P2pal21F, or P1pal7. In yet another embodiment, said composition isinfused into a vascular lumen. In another embodiment, said compositionis introduced by aerosol into the lungs of said subject.

The present invention also provides an inhibitor of platelet activation,comprising a polypeptide comprising an isolated fragment of a nucleotideGPCR and a cell penetrating moiety linked to said polypeptide. In oneembodiment, the inhibitor comprises an isolated fragment of a nucleotideGPCR, wherein the nucleotide receptor is a P2Y₁₂ receptor. In anotherembodiment, the inhibitor comprises a polypeptide wherein saidpolypeptide is SEQ ID NO:33 or SEQ ID NO:34. In yet another embodiment,said inhibitor comprises Y12Pal-18. In another embodiment, saidinhibitor comprises Y12Pal-24.

The present invention also provides a method of inhibiting plateletaggregation, comprising contacting a platelet with a compositioncomprising an isolated fragment of a nucleotide receptor linked to acell penetrating moiety. In one embodiment, the isolated fragment of anucleotide receptor is a P2Y₁₂ receptor.

The present invention also provides a method of inhibiting thrombusformation in a mammal, comprising administering to said mammal acomposition comprising an isolated fragment of a P2Y₁₂ receptor linkedto a cell penetrating moiety. In one embodiment, said composition isinfused into a vascular lumen. In one example, said lumen is a jugularvein or a peripheral vein. In yet another embodiment, said compositionis infused into the lungs of said mammal. In another embodiment, saidcomposition is injected into the peritoneal cavity of said mammal. Inyet another embodiment, said composition is injected subdermally orsubcutaneously into said mammal. In another embodiment, said compositionis administered transdermally to said mammal.

The present invention also provides a vascular endoprosthetic device,comprising an inhibitor of thrombus formation, said inhibitor comprisingan isolated fragment of a nucleotide receptor linked to a cellpenetrating moiety. In one embodiment, said device is a stent. Inanother embodiment, said device is a catheter. In another embodiment,said device is impregnated with said inhibitor. In yet anotherembodiment, said device is coated with said inhibitor.

The present invention also provides a soluble chimeric polypeptidecomprising: a first domain comprising an extracellular or anintracellular fragment of a GPCR; and a second domain comprising acell-penetrating or a membrane-tethering moiety, linked to said firstdomain, wherein said second domain comprises a naturally occurringcontiguous amino acid from a transmembrane domain adjacent to saidextracellular or intracellular fragment, wherein said transmembranedomain is not transmembrane domain 1-7 of the CXCR4, transmembranedomain 1-7 of the CCKA receptor, or transmembrane 2 of the CCR5receptor.

The present invention also provides a soluble chimeric polypeptidecomprising: a first domain comprising an extracellular or anintracellular fragment of a GPCR; and a second domain comprising acell-penetrating or a membrane-tethering moiety, linked to said firstdomain, wherein said second domain comprises 1-15 contiguous amino acidsof a naturally-occurring transmembrane helix domain adjacent to saidextracellular or intracellular fragment.

The present invention also provides a composition comprising apolypeptide, said polypeptide comprising an isolated intracellular orextracellular fragment of a G-protein coupled receptor (GPCR); and anon-peptidic cell penetrating, membrane tethering moiety linked to saidpolypeptide. In one embodiment, the compositions according to thepresent invention comprises cell penetrating moiety comprising at least10 contiguous amino acids of a GPCR transmembrane helix domain. Inanother embodiment, the compositions according to the present inventioncomprises cell penetrating moiety comprises a C₉-C₂₄ fatty acid. In yetanother embodiment, the compositions according to the instant inventioncomprises membrane-tethering moiety comprises 1-7 contiguous amino acidsof a GPCR transmembrane helix domain. In one embodiment, saidmembrane-tethering moiety comprises the amino acid sequence YCYVSII (SEQID NO: 40). In another embodiment, said membrane-tethering moiety isselected from the group consisting of a C₁ acyl group, a C₂ acyl group,a C₃ fatty acid, a C₄ fatty acid, a C₃ fatty acid, a C₆ fatty acid, a C₇fatty acid, and a C₈ fatty acid.

The present invention also provides chimeric polypeptide comprising afirst domain comprising extracellular or intracellular portions of a Gprotein coupled receptor, and at least a second domain, attached to thefirst domain, wherein said second domain is a naturally or non-naturallyoccurring hydrophobic moiety, and wherein said first domain does notcomprise a native extracellular ligand of said GPCR. In one embodimentof the chimeric polypeptide of the invention, the chimeric polypeptidethe second domain or other domains are attached at either one end, atboth ends, or at an internal position of said first domain.

In another embodiment of the chimeric polypeptide of the invention, thehydrophobic moiety is a lipid, an acyl or an amino acid. In yet anotherembodiment of the chimeric polypeptide of the invention, the hydrophobicmoiety is selected from the group consisting of: phospholipids;steroids; sphingosines; ceramides; octyl-glycine; 2-cyclohexylalanine;benzolylphenylalanine; propionoyl (C₃); butanoyl (C₄); pentanoyl (C₅);caproyl (C₆); heptanoyl (C₇); capryloyl (C₈); nonanoyl (C₉); capryl(C₁₀); undecanoyl (C₁₁); lauroyl (C₁₂); tridecanoyl (C₁₃); myristoyl(C₁₄); pentadecanoyl (C₁₅); palmitoyl (C₁₆); phtanoyl ((CH₃)₄);heptadecanoyl (C₁₇); stearoyl (C₁₅); nonadecanoyl (C₁₉); arachidoyl(C₂₀); heniecosanoyl (C₂₁); behenoyl (C₂₂); trucisanoyl (C₂₃); andlignoceroyl (C₂₄); wherein said hydrophobic moiety is attached to saidchimeric polypeptide with amide bonds, sulfhydryls, amines, alcohols,phenolic groups, or carbon-carbon bonds. In another embodiment, thehydrophobic moiety is a transmembrane domain of the GPCR or a fragmentthereof.

In yet another embodiment of the chimeric polypeptide of the invention,the hydrophobic moiety is palmitate (C₁₆), myristoyl (C₁₂), capryl(C₁₀), caproyl (C₆), phospholipids, steroids, sphingosines, ceramides,octyl-glycine, 2-cyclohexylalanine, or benzolylphenylalanine, whereinthe hydrophobic moiety is attached to chimeric polypeptide with amidebonds, sulfhydryls, amines, alcohols, phenolic groups, or carbon-carbonbonds.

In another embodiment of the chimeric polypeptide of the invention, thechimeric polypeptide comprises an extracellular portion selected fromthe group consisting of the first extracellular domain or a fragmentthereof, the second extracellular loop or a fragment thereof, the thirdextracellular loop or a fragment thereof, and the fourth extracellularloop or a fragment thereof, of said G-protein coupled receptor.

In yet another embodiment of the chimeric polypeptide of the invention,the intracellular portion is selected from the group consisting of thefirst intracellular loop or a fragment thereof, the second intracellularloop or a fragment thereof, the third intracellular loop or a fragmentthereof, and the fourth intracellular domain or a fragment thereof, ofsaid G-protein coupled receptor.

In another embodiment of the chimeric polypeptide of the invention, theintracellular portion is selected from the group consisting of anintracellular domain of a one-transmembrane domain G-protein coupledreceptor of the cytokine GPCR or a fragment thereof and an intracellulardomain of a multi-polypeptide-GPCR. In yet another embodiment, themulti-polypeptide-GPCRs is selected from the group consisting of aGPIbNIIX receptor and a collagen receptor.

In another embodiment of the chimeric polypeptide of the invention, theextracellular portion of the GPCR has at least 3 contiguous amino acidresidues. In another embodiment, the intracellular portion has at least3 contiguous amino acid residues or at least 5 contiguous amino acidresidues. In yet another embodiment, said intracellular portioncomprises the third intracellular loop of the GPCR. In anotherembodiment, said intracellular portion comprises at least 7 contiguousamino acid residues of the third intracellular loop.

In another embodiment of the chimeric polypeptide of the invention, thesecond domain of the chimeric polypeptide comprises a GPCR transmembranedomain or a fragment thereof. In another embodiment, the chimericpolypeptide comprises a transmembrane domain with at least 7 amino acidresidues of TM5 or at least 14 amino acid residues of TM5. In anotherembodiment, the chimeric polypeptide comprises amino acid residues whichare contiguous amino acid residues of TM5.

In another embodiment of the chimeric polypeptide of the invention, theG-protein coupled receptor involved is a mammalian G-protein coupledreceptor. In some embodiments, the G-protein coupled receptor orfragment thereof, is selected from the group consisting of a luteinizinghormone receptor, a follicle stimulating hormone receptor, a thyroidstimulating hormone receptor, a calcitonin receptor, a glucagonreceptor, a glucagon-like peptide 1 receptor (GLP-1), a metabotropicglutamate receptor, a parathyroid hormone receptor, a vasoactiveintestinal peptide receptor, a secretin receptor, a growth hormonereleasing factor (GRF) receptor, protease-activated receptors (PARs),cholecystokinin receptors, somatostatin receptors, melanocortinreceptors, ADP receptors, adenosine receptors, thromboxane receptors,platelet activating factor receptor, adrenergic receptors, 5-HTreceptors, CXCR4, CCR5, chemokine receptors, neuropeptide receptors,opioid receptors, erythropoietin receptor, von Willebrand receptor,parathyroid hormone (PTH) receptor, vasoactive intestinal peptide (VIP)receptor, and collagen receptors.

In another embodiment of the chimeric polypeptide of the invention, thehydrophobic moiety is a lipid. In one aspect of this embodiment, thelipid is a palmitate lipid.

The present invention also provides a nucleic acid encoding a chimericpolypeptide comprising a first domain comprising extracellular orintracellular portions of a G protein coupled receptor, and at least asecond domain, attached to the first domain, wherein said second domainis naturally or non-naturally occurring hydrophobic moieties, andwherein said first domain does not comprise a native extracellularligand of said GPCR. The present invention also provides a recombinantvector comprising said nucleic acids encoding chimeric polypeptide and ahost cell transformed with the said recombinant vector.

The present invention also provides a method for producing a chimericpolypeptide comprising cultivating the host cell transformed with therecombinant vector comprising a nucleic acid encoding chimericpolypeptide under conditions sufficient to express the GPCR receptor.

The present invention also provides a method for identifying a potentialtherapeutic agent for use in treatment of a pathology, wherein thepathology is related to aberrant expression or aberrant physiologicalinteractions of a GPCR, comprising providing a cell having a GPCR or aproperty or function ascribable to said GPCR; contacting the cell with acomposition comprising a candidate substance and further contacting thecell with a composition comprising the chimeric polypeptide, anddetermining whether the composition comprising the candidate substancealters the property or function ascribable to said GPCR, whereby, if analteration observed in the presence of the substance is not observedwhen the cell is contacted with a composition devoid of the substance,the substance is identified as a potential therapeutic agent.

The present invention also provides a method of treating or preventing apathology associated with a GPCR, comprising administering the chimericpolypeptide to a subject in which such treatment or prevention isdesired in an amount sufficient to treat or prevent said pathology insaid subject. In one embodiment, the subject is a human.

The present invention also provides a pharmaceutical compositioncomprising the chimeric polypeptide and a pharmaceutically acceptablecarrier. In one embodiment, a pharmaceutical composition comprises thenucleic acid encoding the chimeric polypeptide and a pharmaceuticallyacceptable carrier.

The present invention also provides a kit comprising in one or morecontainers, the pharmaceutical composition comprising the chimericpolypeptide and a pharmaceutically acceptable carrier.

The present invention also provides the use of a therapeutic in themanufacture of a medicament for treating a syndrome associated with ahuman disease, the disease selected from a pathology associated with thechimeric polypeptide wherein said therapeutic is the chimericpolypeptide comprising a first domain comprising extracellular orintracellular portions of a G protein coupled receptor; and at least asecond domain, attached to the first domain, wherein said second domainis naturally or non-naturally occurring hydrophobic moieties, andwherein said first domain does not comprise a native extracellularligand of said GPCR.

The present invention also provides a method for screening for amodulator of activity of a GPCR comprising administering a test compoundto a first test animal, wherein the test animal expresses a desiredGPCR, administering a chimeric polypeptide to a second test animal,measuring the activity of said test compound in said first test animaland said polypeptide in said second test animal; and comparing theactivity of said polypeptide in said second test animal with theactivity of said test compound in said first test animal with theactivity of the desired GPCR in a control animal not administered saidpolypeptide, wherein a change in the activity of said polypeptide insaid first test animal relative to both said second test animal and saidcontrol animal indicates the test compound is a modulator of, an agonistof or an antagonist of said GPCR.

The present invention also includes a method of treating a pathologicalstate in a mammal, the method comprising administering to the mammal achimeric polypeptide comprising a first domain comprising extracellularor intracellular portions of a G protein coupled receptor, and at leasta second domain, attached to the first domain, wherein said seconddomain is naturally or non-naturally occurring hydrophobic moieties, andwherein said first domain does not comprise a native extracellularligand of said GPCR.

As used herein, a “fragment of a GPCR” means a peptide having a portionof the sequence of a GPCR protein which is less than the entirenaturally-occurring amino acid sequence of the GPCR. An “isolatedfragment of a GPCR” means a peptide having a portion of the sequence ofa GPCR protein which is less than the entire sequence, and does notcontain the naturally occurring flanking regions. An isolated GPCRfragment lacks one or more amino acids, which immediately flank thereference fragment in the naturally-occurring molecule. For example, anisolated fragment containing transmembrane region 5 of a reference GPCRsequence lacks at least one amino acid immediately flanking the aminoterminus or carboxyterminus of transmembrane 5.

An “isolated intracellular fragment of a GPCR” means a peptide having anamino acid sequence of an intracellular loop of a GPCR protein, and doesnot contain a sequence from an extracellular loop or a transmembranehelix sequence flanking the intracellular loop. An “isolatedextracellular fragment of a GPCR” means a peptide having an amino acidsequence of an extracellular loop of a GPCR protein and does not containan amino acid of an intracellular loop or transmembrane sequenceflanking regions of the extracellular loop.

An “isolated intracellular fragment of a GPCR” means a peptide having anamino acid sequence of an intracellular loop of a GPCR protein, and doesnot contain a sequence from an extracellular loop or a transmembranehelix sequence flanking the intracellular loop. An “isolatedextracellular fragment of a GPCR” means a peptide having an amino acidsequence of an extracellular loop of a GPCR protein and does not containan amino acid of an intracellular loop or transmembrane sequenceflanking regions of the extracellular loop.

The invention also encompasses compositions containing an isolatedtransmembrane helix fragment or a hybrid transmembrane—intracellularloop or transmembrane—extracellular loop fragment linked to amembrane-tethering or a cell-penetrating moiety. An isolatedtransmembrane helix fragment is a peptide having an amino acid sequenceof one or more contiguous amino acids, but less than all of the aminoacids of a naturally-occurring GPCR transmembrane helix. Atransmembrane-intracellular loop fragment contains at least 3 contiguousamino acids of a naturally-occurring intracellular loop and one or morecontiguous amino acids from one or both flanking GPCR transmembranedomain(s). Preferably, a junctional residue an arginine (R), tryptophan(W) or lysine (K). A junctional residue that is located at a transitionposition between a hydrophobic transmembrane helix domain residues and ahydrophilic intracellular or extracellular loop domain.

In some cases, this junctional residue is considered to terminate thetransmembrane helix. For example, P1pal7 contains 3 amino acids of anintracellular loop (KKS) and RALF (SEQ ID NO:41) from the adjacenttransmembrane domain. The ‘R’ is a junctional residue. In anotherexample, P1pal19 (RCLSSSAVANRSKKSRALF) contains 14 intracellular loopresidues (CLSSSAVANRSKKS), flanked by junctional arginines ‘R’ at boththe N- and C-termini and terminating with ALF from the adjacenttransmembrane domain 6.

As used herein “linked” means attached. For example, a peptide and acell-penetrating (or membrane-tethering) moiety are attached to eachother via a linkage. The linkage is a covalent bond. Preferably, thelinkage is a labile bond, such as a thiol linkage or an ester linkage.One advantage of compounds with a labile linkage is reduced accumulationin body tissues, as compared to compounds with a non-labile linkage.Reduced accumulation in bodily tissues following administration to asubject is associated with decreased adverse side effects in thesubject.

In addition to peptide-based pepducins, the invention encompassescompositions in which the GPCR fragment contains a peptidomimetic. Forexample, the invention includes pepducin compounds in which one or morepeptide bonds have been replaced with an alternative type of covalentbond, which is not susceptible to cleavage by peptidases (a “peptidemimetic” or “peptidomimetic”). Where proteolytic degradation of peptidesfollowing injection into the subject is a problem, replacement of aparticularly sensitive peptide bond with a noncleavable peptide mimeticrenders the resulting peptide more stable and thus more useful as atherapeutic. Such mimetics, and methods of incorporating them intopeptides, are well known in the art. Similarly, the replacement of anL-amino acid residue (e.g., with a D-amino acid) renders the peptideless sensitive to proteolysis.

Additionally, pepducin compounds of the invention can be synthesized asretro-inverso isomers, which include peptides of reverse sequence andchirality. Jameson et al., Nature, 368:744-746 (1994) and Brady et al.,Nature, 368:692-693 (1994). The net result of combining D-enantiomersand reverse synthesis is that the positions of carbonyl and amino groupsin each amide bond are exchanged, while the position of the side-chaingroups at each alpha carbon is preserved. For example, if the peptidemodel is a peptide formed of L-amino acids having the sequence ABC, theretro-inverso peptide analog formed of D-amino acids would have thesequence CBA. The procedures for synthesizing a chain of D-amino acidsto form the retro-inverso peptides are known in the art.

Also useful are amino-terminal blocking groups such ast-butyloxycarbonyl, acetyl, theyl, succinyl, methoxysuccinyl, suberyl,adipyl, azelayl, dansyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl,methoxyazelayl, methoxyadipyl, methoxysuberyl, and 2,4,-dinitrophenyl.Blocking the charged amino- and carboxy-termini of the peptides wouldhave the additional benefit of enhancing passage of the peptide throughthe hydrophobic cellular membrane and into the cell.

An isolated GPCR fragment is derived from the sequence of a Class A GPCRor a Class B GPCR. The isolated GPCR fragment is a fragment from anyknown or unknown GPCR, including, but not limited to protease activatedreceptors (PARs, e.g., a thrombin receptor), a luteinizing hormonereceptor, a follicle stimulating hormone receptor, a thyroid stimulatinghormone receptor, a calcitonin receptor, a glucagon receptor, aglucagon-like peptide 1 receptor (GLP-1), a metabotropic glutamatereceptor, a parathyroid hormone receptor, a vasoactive intestinalpeptide (VIP) receptor, a secretin receptor, a growth hormone releasingfactor (GRF) receptor, cholecystokinin receptors, somatostatinreceptors, melanocortin receptors, nucleotide (e.g., ADP receptors),adenosine receptors, thromboxane receptors, platelet activating factorreceptors, adrenergic receptors, 5-hydroxytryptamine (5-HT) receptors,chemokine receptors (e.g., CXCR4, CCR5), neuropeptide receptors, opioidreceptors, erythropoietin receptor, and parathyroid hormone (PTH)receptor.

In preferred embodiments, the GPCR is a protease-activated receptor, apeptide receptor, or a nucleotide receptor. In particular embodiments,the GPCR is a PAR1, PAR2, PAR3, or a PAR4 receptor. In otherembodiments, the GPCR is a glucagon-like receptor, a nucleotidereceptor, such as a P2Y-₁₂ ADP receptor, a MC4 obesity receptor, a CXCRreceptor (e.g., CXCR4) or CCR5 chemokine receptors, CCKA, or CCKB.

An isolated GPCR fragment includes a fragment of a GPCR which is lessthan 50 contiguous amino acid from the GPCR, and does not contain thenative extracellular ligand of the GPCR. For example, the fragmentcontains between 3 and 30 contiguous amino acids of a GPCR. In preferredembodiments, the GPCR fragment comprises a fragment of a GPCR which isbetween 7 and 24 (inclusive) contiguous amino acids. For example, thefragment includes 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, or 24 contiguous amino acids of a GPCR.

Optionally, the amino acid sequence of a GPCR differs from anaturally-occurring amino acid sequence. For example, individualresidues from a given domain, e.g., a transmembrane helix,extracellular, or intracellular loop, are mutated or substituted with amodified amino acid(s) to improve activity of the pepducin. Preferably,the amino acid sequence of such a GPCR analog differs solely byconservative amino acid substitutions, i.e., substitution of one aminoacid for another of the same class, or by non-conservativesubstitutions, deletions, or insertions located at positions that do notdestroy the function of the protein.

As used herein, a “cell-penetrating moiety” is a compound which mediatestransfer of a substance from an extracellular space to an intracellularcompartment of a cell. Cell-penetrating moieties shuttle a linkedsubstance (e.g., a GPCR peptide) into the cytoplasm or to thecytoplasmic space of the cell membrane. For example, a cell penetratingmoiety is a hydrophobic moiety. The hydrophobic moiety is, e.g., a mixedsequence peptide or a homopolymer peptide (e.g., polyleucine orpolyarginine) which is at least 11 amino acids long. For example, thesubstance is a peptide such as a GPCR fragment or peptidomimetic. Thecell penetrating moiety includes at least 10 contiguous amino acidse.g., 1-15 amino acids of a GPCR transmembrane helix domain. Inparticular embodiments, the hydrophobic moiety is a portion of a GPCR,such as a transmembrane region of a GPCR, e.g., transmembrane region 5(TMR5) of a GPCR.

Cell-penetrating moieties include a lipid, cholesterol, a phospholipid,steroid, sphingosine, ceramide, or a fatty acid moiety. The fatty acidmoiety can be, e.g., any fatty acid which contains at least eightcarbons. For example, the fatty acid can be, e.g., a nonanoyl (C₉);capryl (C₁₀); undecanoyl (C₁); lauroyl (C₁₂); tridecanoyl (C₁₃);myristoyl (C₁₄); pentadecanoyl (C₁₅); palmitoyl (C₁₆); phytanoyl (methylsubstituted C₁₆); heptadecanoyl (C₁₇); stearoyl (C₁₈); nonadecanoyl(C₁₉); arachidoyl (C₂₀); heniecosanoyl (C₂₁); behenoyl (C₂₂);trucisanoyl (C₂₃); or a lignoceroyl (C₂₄) moiety. The cell-penetratingmoiety can also include multimers (e.g., a composition containing morethan one unit) of octyl-glycine, 2-cyclohexylalanine, orbenzolylphenylalanine. The cell-penetrating moiety contains anunsubstituted or a halogen-substituted (e.g., chloro) biphenyl moiety.Substituted biphenyls are associated with reduced accumulation in bodytissues, as compared to compounds with a non-substituted biphenyl.Reduced accumulation in bodily tissues following administration to asubject is associated with decreased adverse side effects in thesubject.

Preferably, the cell penetrating moiety is a naturally-occurring ornon-naturally occurring palmitoyl moiety. For example, the compositionof the invention includes an intracellular loop 4 (i4) of a GPCR and acys-palmitoyl moiety, e.g., the pepducin includes the i4 loop of PAR 1or PAR4 linked to a palmitoyl moiety.

The cell-penetrating moiety is attached to the C-terminal amino acid,the N-terminal amino acid, or to an amino acid between the N-terminaland C-terminal amino acid of the GPCR fragment.

As used herein, a “membrane-tethering moiety” is a compound whichassociates with or binds to a cell membrane. Thus, themembrane-tethering moiety brings the substance to which themembrane-tethering moiety is attached in close proximity to the membraneof a target cell. The substance is a peptide such as a GPCR fragment orpeptidomimetic. The cell membrane is a eucaryotic of procaryoticmembrane. The membrane-tethering moiety, for example, is a hydrophobicmoiety. The hydrophobic moiety can be, e.g., a mixed sequence peptide ora homopolymer peptide (e.g., polyleucine or polyarginine) which is lessthan 10 amino acids long. The membrane-tethering moiety can include atleast 1-7 contiguous amino acids of a GPCR transmembrane helix domain,e.g., the membrane-tethering moiety includes the amino acid sequenceVCYVSII (SEQ ID NO: 40), shown in FIG. 1A as P1-i3-26. Preferably, themembrane-tethering moiety is at least 10 contiguous amino acids (butless than 16 amino acids) of a GPCR transmembrane domain; morepreferably, the membrane-tethering moiety is at least 15 contiguousamino acids of a GPCR transmembrane domain. Membrane-tethering moietiesalso include cholesterol, a phospholipid, steroid, sphingosine,ceramide, octyl-glycine, 2-cyclohexylalanine, benzolylphenylalanine.Other membrane-tethering moieties include a C₁ or C₂ acyl group, and aC₃-C₈ fatty acid moiety such as propionoyl (C₃); butanoyl (C₄);pentanoyl (C₅); caproyl (C₆); heptanoyl (C₇); and capryloyl (C₈).

Similar to the cell-penetrating moiety, the membrane-tethering moiety isattached to the C-terminal amino acid, the N-terminal amino acid, or toan amino acid between the N-terminal and C-terminal amino acid of theGPCR fragment in the pepducin.

Also within the invention is a composition which includes a polypeptidehaving an amino acid sequence of SEQ ID NOS: 1-16, SEQ ID NOS:27-29, orSEQ ID NOS 33-36 linked to a cell penetrating moiety. Preferably, thecell-penetrating moiety is a palmitoyl group.

Another aspect of the invention relates to compositions according toFormula I:

A-X-B-Y_(n)  (1),

wherein A is a cell penetrating moiety, X is a linking moiety, B is anisolated intracellular fragment or an isolated extracellular fragment ofa G-protein coupled receptor (GPCR), Y is a hydrophobic peptide or alipid, and n is zero or one. In some embodiments, X is a covalent bondbetween A and B, polyglycine, polyarginine, a mixed sequence hydrophobicpeptide, amino acid, modified amino acid or a small molecule organic(i.e., aromatic) moiety. The covalent bond can be a labile covalent bondsuch as a thiol linkage or an ester linkage.

In various embodiments, the cell-penetrating moiety, A, is attached tothe C-terminal amino acid, the N-terminal amino acid, or to an aminoacid between the N-terminal and C-terminal amino acid of the GPCRfragment in the pepducin.

The compositions are used to treat, prevent, or ameliorate (reduce theseverity of) one or more symptoms associated with diseases andconditions characterized by aberrant GPCR activity. Such diseases andconditions include thrombosis, heart attack, stroke, excessive bleeding,asthma, inflammation, pain, inflammatory pain, visceral pain, neurogenicpain, arthritis, diabetes, HIV infection, anxiety, depression, pulmonaryinsufficiency, and various types of cancer. Such methods are carried outby contacting a cell, which pathologically overexpresses a GPCR with apepducin GPCR antagonist. For example, the method involves administeringto a subject, e.g., a human patient, in which such treatment orprevention is desired a pepducin in an amount sufficient to reduce theseverity of the pathology in the subject. The present invention alsoincludes pharmaceutical compositions containing any of the pepducincompositions and a pharmaceutically acceptable carrier. The inventionalso includes kits containing the pharmaceutical compositions. Theinvention further includes methods of treating a pathological state in amammal through the administration of any polypeptide of the invention.

The invention includes an inhibitor of platelet activation. Theinhibitor contains an isolated fragment of a protease activated receptorand a cell penetrating moiety linked to the GPCR polypeptide. In someembodiments, the protease activated receptor is a thrombin receptor, atrypsin receptor, clotting factor Xa receptor, activated protein Creceptor, tryptase receptor, or a plasmin receptor. The thrombinreceptor is preferably PAR-4 or PAR-1. The isolated fragment of athrombin receptor is preferably SEQ ID NO: 29 or SEQ ID NO: 4. Forexample, the inhibitor of platelet activation includes P4pal10 orP1pal12.

The invention also includes a method of inhibiting platelet aggregation,by contacting a platelet with a composition of an isolated fragment of aprotease activated receptor linked to a cell penetrating moiety asdescribed above. For example, the protease activated receptor is athrombin receptor, such as a PAR-1 receptor or a PAR-4 receptor. Alsowithin the invention is a method of inhibiting thrombus formation in amammal by administering to the mammal a composition of the inventionwhich includes an isolated fragment of a thrombin receptor linked to acell penetrating moiety.

The methods of the invention are carried out by infusing into a vascularlumen, e.g., a jugular vein, peripheral vein or the perivascular space,the inhibitory compositions of the invention. The peripheral vein canbe, e.g., a vein located in the extremities, such as the hand, wrist, orfoot. In some embodiments, the composition is infused into the lungs ofsaid mammal, e.g., as an aerosol. In other embodiments, the compositionof the invention is administered by injection. In various embodiments,the injection can be into the peritoneal cavity of said mammal,subdermally, or subcutaneously. The composition of the invention canalso be administered transdermally. In other embodiments, thecomposition of the invention is administered vaginally or rectally. Thecomposition can be administered by implanting wound packing material ora suppository which is coated or impregnated with the composition of theinvention.

Inhibitors of clot formation or platelet aggregation are used in medicaldevices, e.g., as coatings. For example, a vascular endoprostheticdevice, e.g., a screen, stent or catheter, includes an inhibitor ofthrombus formation which is an isolated fragment of a thrombin receptorlinked to a cell penetrating moiety. The composition is impregnated inthe device and diffuses into bodily tissues upon contact with a tissueor implantation of the device; alternatively, the device is coated withthe pepducin.

Pepducins are also used to inhibit migration and invasion of a tumorcell by contacting the tumor cell with an isolated fragment of aprotease activated receptor linked to a cell penetrating moiety. Theprotease activated receptor is a PAR-4, PAR-2, or a PAR-1 receptor,e.g., a receptor, which includes the amino acid sequence of SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:29. In otherembodiments, the composition comprises P4pal10, P1pal12, P2pal21,P2pal21F. or P1pal7. Methods of inhibiting metastases of a tumor cellare carried out by contacting the tumor cell with an isolated fragmentof a protease activated receptor linked to a cell-penetrating moiety.The tumor cell is a melanoma cell, a breast cancer cell, a renal cancercell, a prostate cancer cell, a lung cancer cell, a colon cancer cell, acentral nervous system (CNS) cancer cell, a liver cancer cell, a stomachcancer cell, a sarcoma cell, a leukemia cell, or a lymphoma cell.

Symptoms of asthma are reduced by administering a thrombin or atrypsin/tryptase GPCR based pepducin. Accordingly, a method ofinhibiting asthma is carried out by administering a compositioncontaining an isolated fragment of a thrombin or a trypsin/tryptase GPCRlinked to a cell penetrating moiety. Preferably, the trypsin/tryptasereceptor is a PAR-1, PAR-2 or PAR-4 receptor. For example, the isolatedfragment of a PAR includes SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQID NO: 8, or SEQ ID NO: 29. In other embodiments, the compositionincludes P4pal10, P1pal12, P2pal21, P2pal21F, or P1pal7. In variousembodiments, the composition is infused into a vascular lumen, such as aperipheral vein, is infused into the lungs of the mammal, e.g., byinhalation (e.g., as an aerosol), or is administered by a transdermalroute.

Inhibitors of platelet activation include an isolated fragment of anucleotide activated GPCR such as a P2Y₁₂ receptor linked to a cellpenetrating moiety. For example, the GPCR polypeptide includes SEQ IDNO:33 or SEQ ID NO:34. In specific embodiments, the composition includesY12Pal-18 or Y12Pal-24. Such compositions are useful in methods ofinhibiting platelet aggregation.

In yet another aspect, the invention includes a method of inhibitingthrombus formation in a mammal by administering a composition includingan isolated fragment of a nucleotide activated receptor linked to a cellpenetrating moiety to the mammal. In some embodiments, the thrombinreceptor is a P2Y₁₂ receptor. The method can be carried out by infusinginto a vascular lumen, e.g., a jugular vein, peripheral vein theinhibitory compositions of the invention. The peripheral vein can be,e.g., a vein located in the extremities, such as the hand, wrist, orfoot. In some embodiments, the composition is infused into the lungs ofsaid mammal, e.g., as an aerosol. In other embodiments, the compositionof the invention is administered by injection. In various embodiments,the injection can be into the peritoneal cavity of said mammal,subdermally, or subcutaneously. In other embodiments, the composition ofthe invention is administered transdermally. The composition can beadministered by implanting wound packing material or a suppository whichis coated or impregnated with the composition of the invention.

In another aspect, the invention includes a vascular endoprostheticdevice, which includes an inhibitor of thrombus formation which is anisolated fragment of a nucleotide receptor linked to a cell penetratingmoiety. In various embodiments, the device can be, e.g., a stent or acatheter. In some embodiments, the device is impregnated with or coatedwith the inhibitor.

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. Other features, objects, and advantages ofthe invention will be apparent from the description. In thespecification and the appended claims, the singular forms also includethe plural unless the context clearly dictates otherwise. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. In the case of conflict, the presentSpecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. All patents and publications cited in this specificationare incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of PAR1.

FIGS. 1B and 1C are line graphs of the fluorescence excitation intensityindicating calcium levels at 340/380 nm.

FIG. 1D is a line graph of light transmittance by platelets.

FIG. 1E are line graphs indicating flow cytometry of Rat1 fibroblasts

FIG. 2A is a table illustrating an alignment of amino acid sequences.

FIGS. 2B-D are line graphs demonstrating the effect of various peptideson PLC-β activity as measured by inositol phosphate formation.

FIGS. 2E-G are bar graphs demonstrating the penetrating ability ofP1pal-19, P1pal-13, P2pal-21, and P2pal21F peptides on COS7 cellstransfected with various receptors, as measured by on PLC-β activity(measured by inositol phosphate formation).

FIGS. 3A-3C are line graphs demonstrating the penetration of pepducinP1pal-19, thrombin, and SFFLRN (SEQ ID NO:23) to activate wild-type (WT)and mutant (C-tail deleted delta377, and S309P) PAR1s, as monitored byPLC-beta-dependent inositol phosphate formation.

FIG. 4A is a line graph of the fluorescence excitation intensityindicating calcium levels at 340/380 nm.

FIG. 4B is a line graph of inhibition of platelet aggregation by variouspeptides of the invention, as measured by light transmittance.

FIG. 4C is a line graph of inhibition of platelet aggregation by variouspeptides of the invention.

FIG. 4D is a line graph demonstrating the effect of various peptides onPLC-13 activity as measured by inositol phosphate formation.

FIG. 4E is a schematic representation of activation ofreceptor-G-protein complexes by pepducins.

FIG. 5 is a bar graph of showing that the peptides of the presentinvention penetrate intact cells.

FIG. 6A is a table of an alignment of various GPCR fragments.

FIGS. 6B-D are line graphs of the activation various receptors bypepducins, as measured by PLC-β activity (measured by inositol phosphateformation).

FIG. 7 is a line graph depicting pepducin activation of the Gs-coupledMC4 obesity receptor.

FIGS. 8A and B are schematic representations of LBS-1 interference ofPAR-1 activation and molecular liganding.

FIGS. 9 A and B are schematic representation of LBS-1 pepducins.

FIG. 9 C is a line graph indicating the fluorescence excitationintensity at 340/380 nm showing that the non-lipidated LBS-1 peptide isa poor antagonist against thrombin and SFLLRN (SEQ ID NO:23) activationof PAR-1-dependent platelet Ca²⁺ fluxes (FIGS. 9C, and 9D,respectively).

FIG. 9E is a line graph showing LBS-1 inhibition of thrombin aggregationof platelets.

FIGS. 10A through 10E depict a schematic of the PAR1 and PAR 4receptors, pepducins of the present invention and their effect on theactivation and/or regulation of Ca²⁺ signaling and aggregation inplatelets. FIG. 10A depicts a schematic of a hypothetical mechanism ofinhibition of PAR1 and PAR4 by their cognate pepducins at theintracellular surface of the plasma membrane. FIGS. 10B and 10C aregraphs indicating the inhibition of aggregation of human platelets bypepducin P1pal12 and P4pal10. FIG. 10D shows fluorescence graphsdemonstrating that preincubation of platelets with P4pal10 attenuatesCa²⁺ signal induction.

FIGS. 11A through 11F show the effect of p4pal-10 on human and mouseplatelets, and an alignment of the third intracellular loops of humanand murine PAR4. FIG. 11A shows a graph illustrating inhibition ofaggregation of human platelets with P1pal12 and P4pal-10 at 3 nMThrombin; FIG. 11B shows a graph illustrating inhibition of aggregationof human platelets with P1pal12 and P4pal-10 at 20 nM Thrombin; FIG. 11Cshows a graph illustrating full platelet aggregation of murine plateletswith P4pal-10 at 1 nM Thrombin; FIG. 11D shows a graph illustrating fullplatelet aggregation of murine platelets with P4pal-10 at 3 nM Thrombin;FIG. 11E shows a graph illustrating full platelet aggregation of murineplatelets with P4pal-10 at 20 nM Thrombin. FIG. 11F shows an alignmentof the third intracellular loops of human and murine PAR4.

FIGS. 12A through 12D indicate that P4pal-10 prolongs bleeding time andprotects against systemic platelet activation. FIG. 12A shows afluorescence graph indicating accumulation of i3 loop peptides incirculating mouse platelets. FIG. 12B is a bar graph depicting unstablehaemostasis as measured by % rebleeding from amputated tail tips in micetreated with pepducin or vehicle alone; FIG. 12C is a graph indicatingtotal tail bleeding times for mice injected with vehicle, P1pal-12 orP4pal-10. FIG. 12D is a bar graph indicating the protective effect ofP4pal10 on systemic platelet activation in mice.

FIGS. 13A and B are graphs showing inhibition of platelet aggregation byP2Y₁₂-based i3 loop pepducins.

FIGS. 14A and B are bar graphs showing activation and inhibition ofglucagon-like peptide-1 receptor (GLP-1R) by GLP-1R-based i3 looppepducins.

FIG. 15 is a bar graph showing the inhibition of chemoinvasion ofMDA-MB-231 breast cancer cells with PAR1 and PAR2-based i3 looppepducins.

FIG. 16 is a graph showing P2Pal-21 inhibition of trypsin activation ofPAR2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the creation of GPCR conjugates whichinclude a GPCR moiety, derived from a GPCR or a fragment thereof, and acell penetrating moiety which partitions the conjugate into and acrossthe lipid bilayer of target cells. The cell penetrating moiety is, e.g.,a hydrophobic region of the GPCR fragment itself. The cell penetrating(or cell surface-associating) moiety anchors the conjugate in the lipidbilayer (or to the cell surface), increasing the effective molarity ofthe conjugate in the vicinity of the intracellular receptor, e.g., atthe receptor-G protein interface. An exogenous GPCR moiety disruptsreceptor-G protein interactions and cause activation and/or inhibitionof signaling. Thus, the methods and compositions, as well as theexperiments detailed herein, demonstrate that selectively targeting theintracellular receptor-G-protein interface using cell-penetratingpeptides results in agonists or antagonists of G-protein receptorsignaling. Specifically, the conjugation of a hydrophobic moiety, suchas a hydrophobic peptide or a moiety containing a long hydrocarbon chainsuch as a palmitoyl group, to peptides derived from a GPCR (e.g., anintracellular loop, such as the third intracellular loop, of PAR1, PAR2,or PAR4), yields full agonists and/or antagonists of G-protein receptorsignaling.

The pepducins are designed to act as receptor-modulating agents bytargeting the intracellular surface of the receptor. Additionalpepducins include PAR1- and PAR4-based antagonists for anti-haemostaticand anti-thrombotic effects under in vivo conditions. Because thrombinis the most potent activator of platelets, PAR1 (Vu et al., Cell 64,1057 (1991)) and PAR4 (Xu et al., Proc. Natl. Acad. Sci. (USA) 95, 6642(1998); Covic et al., Biochemistry 39, 5458 (2000); and Covic et al.,Thromb. Haemost. 87, 722 (2002)) were chosen as targets. Antagonists ofthese two receptors may be useful to prevent the thrombotic andproliferative complications of acute coronary syndromes. Andrade-Gordonet al., J. Pharm. Exp. Therap. 298, 34 (2001) and Ma et al., Br. J.Pharm. 134, 701 (2001).

FIG. 4E shows a two-site mechanism by which pepducins both activate andinhibit receptor-G protein signaling, a two-site mechanism. Themechanism accommodates the biphasic activation and inhibition of theagonists and the inhibition of the antagonists. Pepducins, by virtue oftheir hydrophobic tether, rapidly transduce the plasma membrane andachieve high effective molarity at the perimembranous interface. Thepepducin agonist first occupies a high-affinity site at theintracellular surface of the GPCR. The bound agonist either stabilizesor induces the activated state of the receptor to turn on the associatedG protein(s). After this first site becomes saturated, higherconcentrations of pepducin begin to occupy a second, lower-affinity,inhibitory site that blocks signal transference to G protein in adominant manner, perhaps by mimicking the GPCR (e.g., the receptori3-loop) ground-state interactions with the G protein. The inhibition bythe pepducin antagonists is coincident with the inhibitory phase of theagonists, thus the antagonists may also bind at this lower affinitysite. Exogenous activation or inhibition of receptors by pepducins couldreflect a potential dimerization mode whereby one receptor donates itsintracellular loops to an adjacent receptor. There are several examplesof receptor dimers that give rise to distinct signaling properties(Milligan, Science 288, 65-67 (2000)), including the cytokine/GPCRs suchas the EPO receptor (Guillard et al., J. Biol. Chem. (2001) 276,2007-2013), however, the mechanism(s) of cross-receptor modulation isunknown.

G Protein Coupled Receptors

G protein coupled receptors are intrinsic membrane proteins whichcomprise a large superfamily of receptors. The family of Gprotein-coupled receptors (GPCRs) has at least 250 members (Strader etal. FASEB J., 9:745-754, 1995; Strader et al. Annu. Rev. Biochem.,63:101-32, 1994). It has been estimated that one percent of human genesmay encode GPCRs. Many GPCRs share a common molecular architecture andcommon signaling mechanism. Historically, GPCRs have been classifiedinto six families, originally thought to be unrelated, three of whichare found in vertebrates. Recent work has identified several new GCPRfamilies and suggested the possibility of a common evolutionary originfor all of them.

Many GPCRs share a common structural motif of seven transmembranehelical domains. Some GPCRs, however, do not have seven transmembranehelical domains and instead can be single-spanning transmembranereceptors.

Single spanning GPCRs include receptors for cytokines such aserythropoietin, EGF, insulin, insulin-like growth factors I and II, TGF.

GPCR families include Class A Rhodopsin like, Class B Secretin like,Class C Metabotropic glutamate/pheromone, Class D Fungal pheromone,Class E cAMP receptors (Dictyostelium), and Frizzled/Smoothened family.Putative families include Ocular albinism proteins, Drosophila odorantreceptors, Plant Mlo receptors, Nematode chemoreceptors, and Vomeronasalreceptors (V1R & V3R).

Class A Rhodopsin like receptors include: Amine receptors:Acetylcholine, Alpha Adrenoceptors, Beta Adrenoceptors, Dopamine,Histamine, Serotonin, Octopamine, and Trace amine; Peptide receptors:Angiotensin, Bombesin, Bradykinin, C5a anaphylatoxin, Fmet-leu-phe, APJlike, Interleukin-8, Chemokine receptors (C-C Chemokine, C-X-CChemokine, BONZO receptors (CXC6R), C-X3-C Chemokine, and XC Chemokine),CCK receptors, Endothelin receptors, Melanocortin receptors,Neuropeptide Y receptors, Neurotensin receptors, Opioid receptors,Somatostatin receptors, Tachykinin receptors, (Substance P (NK1),Substance K (NK2), Neuromedin K (NK3), Tachykinin like 1, and Tachykininlike 2), Vasopressin-like receptors (Vasopressin, Oxytocin, andConopressin), Galanin like receptors (Galanin, Allatostatin, and GPCR54), Proteinase-activated like receptors (e.g., Thrombin), Orexin &neuropeptide FF, Urotensin II receptors, Adrenomedullin (G10D)receptors. GPR37/endothelin B-like receptors, Chemokine receptor-likereceptors, and Neuromedin U receptors; Hormone protein receptors:Follicle stimulating hormone, Lutropin-choriogonadotropic hormone,Thyrotropin, and Gonadotropin; (Rhod)opsin receptors; Olfactoryreceptors; Prostanoid receptors: Prostaglandin, Prostacyctin, andThromboxane; Nucleotide-like receptors: Adenosine and Purinoceptors;Cannabis receptors; Platelet activating factor receptors;Gonadotropin-releasing hormone receptors: Thyrotropin-releasing hormone& Secretagogue receptors: Thyrotropin-releasing hormone, Growth hormonesecretagogue, and Growth hormone secretagogue like; Melatonin receptors;Viral receptors; Lysosphingolipid & LPA (EDG) receptors; Leukotriene B4receptor: Leukotriene B4 receptor BLT1 and Leukotriene B4 receptor BLT2;and Class A Orphan/other receptors: Platelet ADP & KI01 receptors, SREB,Mas proto-oncogene, RDC1, ORPH, LGR like (hormone receptors), GPR, GPR45like, Cysteinyl leukotriene, Mas-related receptors (MRGs), and GP40 likereceptors.

Class B (the secretin-receptor family or ‘family 2’) of the GPCRs is asmaller but structurally and functionally diverse group of proteins thatincludes receptors for polypeptide hormones (Calcitonin, Corticotropinreleasing factor, Gastric inhibitory peptide, Glucagon, Glucagon-likepeptide-1,-2, Growth hormone-releasing hormone, Parathyroid hormone,PACAP, Secretin, Vasoactive intestinal polypeptide, Diuretic hormone,EMR1, Latrophilin), molecules thought to mediate intercellularinteractions at the plasma membrane (Brain-specific angiogenesisinhibitor (BAI)) and a group of Drosophila proteins (Methuselah-likeproteins) that regulate stress responses and longevity.

Class C Metabotropic glutamate/pheromone receptors include Metabotropicglutamate, Metabotropic glutamate group I, Metabotropic glutamate groupII, Metabotropic glutamate group III, Metabotropic glutamate other,Extracellular calcium-sensing, Putative pheromone Receptors, GABA-B,GABA-B subtype 1, GABA-B subtype 2, and Orphan GPRC5 receptors.

GPCRs can potentially be multi-polypeptide receptors such as GPIb-V-IX,or the collagen receptor, that exhibit outside-in-signaling via Gproteins.

Although hundreds of G protein coupled receptor genes or cDNAs have beencloned, it is believed that there are still many uncharacterized Gprotein coupled receptors which have not yet been recognized as GPCRs.

GPCRs play a vital role in the signaling processes that control cellularmetabolism, cell growth and motility, adhesion, inflammation, neuronalsignaling, and blood coagulation. G protein coupled receptor proteinsalso have a very important role as targets for a variety of signalingmolecules which control, regulate, or adjust the functions of livingbodies. The signaling species can be endogenous molecules (e.g.,neurotransmitters or hormones), exogenous molecules (e.g., odorants),or, in the case of visual transduction, light.

For instance, GPCRs include receptors for biogenic amines, e.g.,dopamine, epinephrine, histamine, glutamate (metabotropic effect),acetylcholine (muscarinic effect), and serotonin; receptors for lipidmediators of inflammation such as prostaglandins, platelet activatingfactor, and leukotrienes; receptors for peptide hormones such ascalcitonin, C5a anaphylatoxin, follicle stimulating hormone,gonadotropin releasing hormone, neurokinin, oxytocin; receptors forproteases such as thrombin, trypsin, tryptase, activated protein C, andfactor VIIa/Xa; and receptors for sensory signal mediators, e.g.,retinal photopigments and olfactory stimulatory molecules. Each moleculeis specific to a receptor protein, whereby the specificities ofindividual physiologically active substances (including specific targetcells and organs), specific pharmacological actions, specific actionstrength, action time, etc., are decided. Thus, GPCRs are a major targetfor drug action and development.

Upon ligand binding, GPCRs regulate intracellular signaling pathways byactivating guanine nucleotide-binding proteins (G proteins). The domainstructure of GPCRs are conserved among members of the GPCR family.Domain boundaries of TM helix domains, intracellular loop domains, andextracellular domains of GPCRS are known in the art. The structure ofunmapped GPCRs is determined by comparison to the prototype GPCR,rhodopsin, using known methods, e.g., as described in Palczewski et al.,Science 289:739 (2000), hereby incorporated by reference.

One characteristic feature of most GPCRs is that seven clusters ofhydrophobic amino acid residues, or transmembrane regions (TMs, the 7transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6,and TM7) are located in the primary structure and pass through (span)the cell membrane at each region thereof (FIG. 1A). The domains arebelieved to represent transmembrane alpha-helices connected by threeintracellular loops (i1, i2, and i3), three extracellular loops (e1, e2,and e3), and amino (N)- and carboxyl (C)-terminal domains (Palczewski etal., Science 289, 739-45 (2000)). Most GPCRs have single conservedcysteine residues in each of the first two extracellular loops whichform disulfide bonds that are believed to stabilize functional proteinstructure. It is well known that these structures detailed above arecommon among G protein coupled receptor proteins and that the amino acidsequences corresponding to the area where the protein passes through themembrane (membrane-spanning region or transmembrane region) and theamino acid sequences near the membrane-spanning region are often highlyconserved among the receptors. Thus, due to the high degree of homologyin GPCRs, the identification of novel GPCRs, as well identification ofboth the intracellular and the extracellular portions of such novelmembers, is readily accomplished by those of skill in the art. FIG. 1Ais a schematic representation of PAR1, indicating the topologicalarrangement of the membrane-spanning segments (TM 1-7), extracellularloops (e1-e4), and intracellular loops (i1-i4). This structure is basedon the X-ray structure of rhodopsin (Palczewski et al., Science 289,739-45 (2000)). As shown in this figure, thrombin cleaves theextracellular domain (e1) at the R41-S42 bond creating a new N-terminus,₄₂SFLLRN (SEQ ID NO: 23), which functions as a tethered PAR1 agonist.

The binding sites for small ligands of G-protein coupled receptors arebelieved to comprise a hydrophilic socket located near the extracellularsurface which is formed by several GPCR transmembrane domains. Thehydrophilic socket is surrounded by hydrophobic residues of theG-protein coupled receptors. The hydrophilic side of each G-proteincoupled receptor transmembrane helix is postulated to face inward andform the polar ligand binding site. TM3 has been implicated in severalGPCRs as having a ligand binding site which includes the TM3 aspartateresidue. TM5 serines, a TM6 asparagine and TM6 or TM7 phenylalanines ortyrosines are also implicated in ligand binding. The ligand binding sitefor peptide hormones receptors and receptors with other larger ligandssuch as glycoproteins (e.g., luteinizing hormone, follicle stimulatinghormone, human chorionic gondaotropin, thyroid-stimulating hormone(Thyrotropin)), and the Ca²⁺/glutamate/GABA (gamma-aminobutyric acid)classes of receptors likely reside in the extracellular domains andloops.

A key event for the switch from inactive to active receptor isligand-induced conformational changes of transmembrane helices 3 (TM3)and 6 (TM6) of the GPCRs that have 7 transmembrane spanning helices(Gether and Kolbilka, J. Biol. Chem. 273, 17979-17982 (1998)). Thesehelical movements in turn alter the conformation of the intracellularloops of the receptor to promote activation of associated heterotrimericG proteins. Mutagenesis studies (Cotecchia et al., J. Biol. Chem.267:1633-1639 (1992); Kostenis et al., Biochemistry 36:1487-1495 (1997);Kjelsberg et al., J. Biol. Chem. 267:1430-1433 (1992)) demonstrated thatthe third intracellular loop (i3) mediates a large part of the couplingbetween receptor and G protein. 13 loops expressed as minigenes havealso been shown to directly compete with adrenergic receptors for Gqbinding (Luttrel et al., Science 259: 1453-1457 (1993)), or can activateG proteins as soluble peptides in cell-free conditions (Okamoto et al.,Cell 67, 723-730 (1991)).

One particular class of GPCR is the protease activated receptors (PARs).Protease-activated receptors (PARs) are members of the superfamily ofG-protein-coupled receptors that initiate cell signaling by theproteolytic activity of extracellular serine proteases. PARs areactivated after proteolytic cleavage of the amino terminus of thereceptor by endogenous proteases, including thrombin (PAR-1, -3, and -4)and trypsin/tryptase (PAR-2 and -4). Of these, PAR2 (Nystedt et al.,Proc. Natl. Acad. Sci. (USA) 91:9208-9212 (1994)) is atrypsin/tryptase-activated receptor that is important in inflammationand pain, and PAR4 (Xu et al., Proc. Natl. Acad. Sci. (USA) 95:6642-6646(1998); Kahn et al., Nature (London) 394:690-694 (1998)) is a secondthrombin receptor that plays a unique role in platelet aggregation(Covic et al., Biochemistry 39, 5458-5467 (2000)).

Because both thrombin, trypsin, and tryptase are present in inflamedairways, PARs are likely to play a major role in airway inflammation.Knight et al., J. Allergy Clin. Immunol. 108:797-803 (2001).

In addition to its pivotal role in hemostasis, thrombin activatesvarious cell types such as platelets and vascular smooth muscle cellsvia proteolytic cleavage of specific cell-surface receptors (PARs), theprototype of which is PAR-1. Thrombin receptor activation is likely toplay a key role in cardiovascular disorders such as arterial thrombosis,atherosclerosis and restenosis, and as such a thrombin receptorantagonist should have potential utility in the treatment of thesedisorders. Chackalamannil, Curr. Opin. Drug Discov. Devel. 4:417-27(2001).

Thrombin is thought to be involved in functional loss after injury tothe mammalian central nervous system (CNS). Down-regulation of PAR-1 hasbeen shown to increase post-traumatic survival of CNS neurons andpost-traumatic toxicity of thrombin may be down-regulated by appropriatemodulation of PAR-1 receptors. Friedmann et al., Neuroimmunol.,121:12-21 (2001).

PARS are also involved in a variety of other diseases or indications,including various cancers, cellular proliferation, and pain.

GPCR Domains

Most GPCRs are characterized by seven clusters of hydrophobic amino acidresidues, or transmembrane regions (TMs, the 7 transmembrane regions aredesignated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7), that are locatedin the primary structure and pass through (span) the cell membrane (FIG.1A). The TM regions are believed to represent transmembranealpha-helices connected by three intracellular loops (i1, i2, and i3),three extracellular loops (e1, e2, and e3). GPCRs also contain amino(N)- and carboxyl (C)-terminal domains (Palczewski et al., Science 289,739-45 (2000)). The sequences between the transmembrane regionscorrespond to GPCR loops, and the location of a loop within a celldetermines whether it is an intracellular or an extracellular loop. MostGPCRs have single conserved cysteine residues in each of the first twoextracellular loops which form disulfide bonds that are believed tostabilize functional protein structure. A schematic representation oftransmembrane and loop regions of the PAR1 GPCR is presented in FIG. 1A.

One example of a GPCR is the CXCR4 receptor, shown in Table 1 as SEQ IDNO:38. The seven underlined sequences correspond to the seventransmembrane regions of the GPCR. Thus, the sequenceIFLPTIYSIIFLTGIVGNGLVILV (SEQ ID NO:39) corresponds to the firsttransmembrane region (TM 1).

TABLE 1 CXCR4MEGISIYTSD NYTEEMGSGD YDSMKEPCFR EENANFNKIF LPTIYSIIFL TGIVGNGLVILVMGYQKKLR SMTDKYRLHL SVADLLFVIT LPFWAVDAVA NWYFGNFLCK AVHVIYTVNLYSSVLILAFI SLDRYLAIVH ATNSQRPRKL LAEKVVYVGV WIPALLLTIP DFIFANVSEADDRYICDRFY PNDLWVVVFQ FQHIMVGLIL PGIVILSCYC IIISKLSHSK GHQKRKALKTTVILILAFFA CWLPYYIGIS IDSFILLEII KQGCEFENTV HKWISITEAL AFFHCCLNPILYAFLGAKFK TSAQHALTSV SRGSSLKILS KGKRGGHSSV STESESSSFH SS.(SEQ ID NO: 38)

An isolated fragment of a GPCR is any portion of the GPCR which is lessthan the full length protein. A peptide containing an isolated fragmentof a GPCR may contain an amino acid sequence N-terminal and/orC-terminal to the GPCR sequence other than the naturally occurring aminoacid sequence. A peptide containing an isolated transmembrane sequenceof a GPCR may contain only the sequence corresponding to thattransmembrane region of the GPCR, or it may also contain amino acidsequences N-terminal and/or C-terminal to the transmembrane sequence,that are not the naturally occurring flanking sequences (i.e., not theloop sequences which are adjacent to that region in the naturallyoccurring GPCR sequence).

Thus, a peptide containing an isolated transmembrane region of the CXCR4receptor is any peptide that contains any or all of the contiguous aminoacids of an underlined region of sequence shown in Table 1. Such apeptide does not contain any of the naturally occurring (non-underlined)flanking sequence which corresponds to loop sequences which are adjacentto that TM region in the naturally occurring GPCR sequence.

Likewise, a peptide containing an isolated (intracellular orextracellular) loop region of the CXCR4 receptor is any peptide thatcontains any or all contiguous amino acids of a non-underlined region ofsequence shown in Table 1. Such a peptide does not contain any of thenaturally occurring transmembrane sequences, shown as underlinedflanking sequence in Table 1, which are adjacent to that loop region inthe naturally occurring GPCR sequence.

A peptide containing an isolated extracellular domain or an isolatedintracellular domain can include amino acid sequences from any(extracellular or intracellular) loop and/or the N- or C-terminaldomain. Such a peptide does not include any sequence from atransmembrane region which is adjacent to that extracellular domain orintracellular domain in the naturally occurring GPCR sequence.

Pharmaceutical Compositions

The pepducins (also referred to herein as “active compounds”) of theinvention, and derivatives, fragments, analogs and homologs thereof, canbe incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the pepducin and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Suitable carriers are described in themost recent edition of Remington's Pharmaceutical Sciences, a standardreference text in the field, which is incorporated herein by reference.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous (e.g., aperipheral vein, such as found in the extremities), intraperitoneal,intradermal, subcutaneous, subdermal, oral, intranasal, aerosol (e.g.,inhalation), transdermal (i.e., topical), transmucosal, vaginal,intrauterine, and rectal (e.g., suppositories) administration.Injectable solutions containing active compounds of the presentinvention may be administered to the vascular lumen of vessels (e.g.,aorta or jugular vein). Alternatively, active compounds of the presentinvention may be administered via a device, e.g., stent or catheter,impregnated or coated with the active compounds.

Solutions or suspensions used for administration (e.g., parenteral) mayinclude the following components: a sterile diluent such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents; antibacterial agents suchas benzyl alcohol or methyl parabens; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid (EDTA); buffers such as acetates, citrates or phosphates, andagents for the adjustment of tonicity such as sodium chloride ordextrose. The pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide. A preparation of a pharmaceuticalcomposition of the present invention can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation or intranasal administration, thecompounds are delivered in the form of an aerosol spray from pressuredcontainer or dispenser which contains a suitable propellant, e.g., a gassuch as carbon dioxide, or a nebulizer (e.g., delivery to the lung).

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The active compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides), a suppository coating, or retention enemas for rectaldelivery. The active compounds can be similarly prepared forintravaginal or intrauterine administration. The active compounds mayalso be administered as impregnated in or as a coating on wound packing(e.g., to reduce bleeding).

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pepducins and GPCR peptides can be administered for the treatment ofvarious disorders in the form of pharmaceutical compositions. Principlesand considerations involved in preparing such compositions, as well asguidance in the choice of components are provided, for example, inRemington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R.Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; DrugAbsorption Enhancement: Concepts, Possibilities, Limitations, AndTrends, Harwood Academic Publishers, Langhorne, Pa., 1994; and PeptideAnd Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4),1991, M. Dekker, New York. The formulation herein can also contain morethan one active compound as necessary for the particular indicationbeing treated, preferably those with complementary activities that donot adversely affect each other. Alternatively, or in addition, thecomposition can comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended. Theactive ingredients can also be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacrylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles, and nanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

Controlled release of active compounds can utilize various technologies.Devices, e.g., stents or catheters, are known having a monolithic layeror a coating incorporating a heterogeneous solution and/or dispersion ofan active agent in a polymeric substance, where the diffusion of atherapeutic agent is rate limiting, as the agent diffuses through thepolymer to the polymer-fluid interface and is released into thesurrounding fluid. Active compound may be dissolved or dispersed in asuitable polymeric material, such that additional pores or channels areleft after the material dissolves. A matrix device is generallydiffusion limited as well, but with the channels or other internalgeometry of the device also playing a role in releasing the agent to thefluid. The channels can be pre-existing channels or channels left behindby released agent or other soluble substances.

Erodible or degradable devices typically may have the active compoundsphysically immobilized in the polymer. The active compounds can bedissolved and/or dispersed throughout the polymeric material. Thepolymeric material may be hydrolytically degraded over time throughhydrolysis of labile bonds, allowing the polymer to erode into thefluid, releasing the active agent into the fluid. Hydrophilic polymershave a generally faster rate of erosion relative to hydrophobicpolymers. Hydrophobic polymers are believed to have almost purelysurface diffusion of active agent, having erosion from the surfaceinwards. Hydrophilic polymers are believed to allow water to penetratethe surface of the polymer, allowing hydrolysis of labile bonds beneaththe surface, which can lead to homogeneous or bulk erosion of polymer.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The pepducin approach according to the present invention allows the richdiversity of intracellular receptor structures to be exploited both forgeneration of new therapeutic agents and for delineation of themechanisms of receptor-G protein coupling under in vivo conditions. Thepepducins discovered by this strategy may also prove to be moreselective to the extent that the pepducins primarily target the receptorrather than the G protein. In addition, many receptors have beenidentified by genomic and genetic approaches as being important invarious diseases processes but have no known ligands-so-called orphanreceptors. Pepducin agonists and antagonists can be generated which aretailored to these receptors, and may be useful in determining whichsignaling pathways are activated by the orphan receptor in the contextof its native environment. Thus, in the post-genomic era, the pepducinapproach may be widely applicable to the targeting of membrane proteinsand may open up new experimental avenues in systems previously notamenable to traditional molecular techniques

Example 1 Manufacture and Characterization of Pepducin Compositions

Synthesis by standard Fmoc solid phase synthetic methods and preparationof palmitoylated peptides was performed as previously described. Covicet al., PNAS 99:643-648 (2002). Palmitoylated peptides were purifiedto >95% purity by C₁₈ or C₄ reverse phase chromatography and dissolvedin DMSO.

For human studies, whole blood was drawn from healthy volunteer donorswith an 18 gauge needle into a 30 mL syringe containing 3 mL of 4%sodium citrate (0.4% v/v final). Gel-filtered human platelets wereprepared as previously described. Hsu-Lin, et al., J. Biol. Chem. 259,9121 (1984). Platelet counts were adjusted to 1.5×10⁵/μL in modifiedPIPES buffer and platelet aggregation was carried out in the presence of2.5 mM CaCl₂ in final volumes of 250 μL. Platelet aggregation wasmeasured by light scattering using a Chronolog 560VS/490-2Daggregometer. Cytosolic calcium measurements were performed ongel-filtered human platelets as previously described. Covic et al.,Biochemistry 39, 5458 (2000). For mouse platelet studies, whole blood(0.5-1 mL/mouse) was collected from the inferior vena cava intoheparinized syringes and pooled from 5 mice. Washed platelets wereprepared by centrifugation of platelet-rich plasma containing 0.1 U/mlof apyrase/1 mM EDTA and aggregation studies conducted as previouslydescribed. Azam et al., Mol. Cell. Biol. 21, 2213 (2001).

Bleeding times were performed with 6-8 week-old adult male CF-1 miceanaesthetized with an intraperitoneal injection of xylazine (10 mg/kg)plus ketamine (50 mg/kg). The internal jugular vein was cannulated witha 0.28×1.52 mm gauge catheter and P1pal-12 (3 μmoles/L), P4pal-10 (3μmoles/L) or vehicle alone (DMSO), was infused over 1 min in a totalvolume of 100 μL. Experiments were performed blind to injectedsubstance. After 5 min, tails were amputated 2 mm from the tail tip.Tails were immersed in a beaker of phosphate-buffered saline maintainedat 37° C. and bleeding was visually followed and timed. If bleedingrestarted within 5 min, it was recorded as a re-bleed and taken to markan unstable haemostasis event as previously described. Law et al.,Nature 401, 808 (1999). Maximum bleeding time allowed was 10 min afterwhich the tail was cauterized.

Mice were anaesthetized and then cathetherized via jugular vein andinjected with either vehicle alone (DMSO), or 0.3, 1 or 3 μmoles/L ofP4pal-10 in 100 μL volumes over 1 min. After 5 min, systemic plateletactivation was induced with a cocktail of 200 μM AYPGKF (SEQ ID NO:26)plus 5 μM epinephrine. After an additional 5 min following the AYPGKF(SEQ ID NO:26)/epinephrine infusion, blood was collected from theinferior vena cava into heparinized syringes. Blood was diluted two-foldinto Tyrode's buffer containing 0.1 U apyrase/1 mM EDTA. Platelet countin whole blood was determined using a Coulter Counter.

Platelet aggregation is measured spectometrically, using an aggregometersuch as an optical aggregometer, a whole blood aggregometer or anintracellular ionized calcium aggregometer. Platelet activation bydegranulation is measured as an increase of cytoplasmic Ca²⁺, or theappearance of cell surface markers such PAC-1 and CD62P.

Ca²⁺ measurements were performed as described (Kuliopulos et al.,Biochemistry 38, 4572-4585 (1999)). Intracellular Ca²⁺ concentration wasmonitored as the ratio of fluorescence excitation intensity at 340/380nm.

An i3 peptide, designated P1-i3-40, was constructed containing theadjacent transmembrane alpha-helical amino acids from the TM5 of PAR1.As a primary screen for biological activity, the ability of P1-i3-40 wastested for it's ability to stimulate platelet activation by monitoringintracellular Ca²⁺. The composition of the P1 peptides are shown in FIG.1.

When added to platelets, the P1-i3-40 peptide causes a rapidintracellular Ca²⁺ transient (Ca^(2+i)) that mimics the Ca^(2+i)response generated by thrombin (FIG. 1B). The Ca^(2+i) transient has nomeasurable lag phase (<5 s) and the maximum Ca^(2+i) is saturable. Aseries of progressively truncated versions of P1-i3-40 were then made inorder to determine whether the N-terminal hydrophobic region wasrequired for activity. The P1-i3-19 peptide, which completely lackshydrophobic N-terminal residues, causes little stimulation of Ca²⁺fluxes (FIG. 1B). The P1-i3-26 peptide with seven N-terminal hydrophobicresidues, which would be expected to partition to only the outsideleaflet of the lipid bilayer, gives a minor, unregulated Ca^(2+i)response. In contrast, the P1-i3-33 peptide has similar potency to theP1-i3-40 peptide demonstrating that 14 hydrophobic amino acid residuesconfer full in vivo activity to the i3 intracellular loop. Studies withshort membrane-translocating sequences have shown that 11-12 hydrophobicamino acid residues are sufficient to transfer proteins (15-120 kDa)into intact cells (Rojas et al., Nat. Biotech. 16, 370-375 (1998)) andtissues of mice (Schwarze et al., Science 285, 156-159 (1999)).

A palmitate lipid (C₁₆H₃₁O) was added to the GPCR moiety, and TMresidues were removed in order to drastically reduce the size of the i3peptides. Palmitoylated peptides were synthesized by standard Fmoc solidphase synthetic methods with C-terminal amides. Palmitic acid wasdissolved in 50% N-methyl pyrolidone/50% methylene chloride and coupledovernight to the deprotected N-terminal amine of the peptide. Aftercleavage from the resin, palmitoylated peptides were purified to >95%purity by C18 or C4 reverse phase chromatography in DMSO. The palmitoylfunctionality is represented as Pal, and the constructC₁₅H₃₁CONH-Peptide-NH₂ is represented herein as Pal-Peptide.

As shown in FIG. 1B, the palmitoylated i3 loop peptide, P1pal-19 causesa rapid Ca^(2+i) transient that is identical in profile to that causedby the extracellular PAR1 ligand, SFLLRN (SEQ ID NO:23). In addition,P1pal-19 fully activates platelet aggregation (FIG. 1D) with an EC50 of8±3 micromolar. Individual aggregation traces of platelets stimulatedwith 10 Micromolar of indicated peptides or palmitic acid and plateletaggregation was monitored as % light transmittance of stirred plateletsat 37° C. as described. Covic et al., Biochemistry 39, 5458-5467 (2000).P1pal-19 completely inhibits the subsequent Ca^(2+i) response to 30micromolar SFLLRN (SEQ ID NO:23) (FIG. 1C) due to desensitization ofPAR1. Similarly, prestimulation with SFLLRN (SEQ ID) NO:23) completelydesensitizes the platelets to P pal-19. Palmitic acid by itself has noeffect on Ca^(2+i) and platelet aggregation (FIG. 1B, D).

To directly determine whether palmitoylation conferred cell-penetratingabilities, Pt-i3-19 and P1pal-19 were labeled with fluorescein (Fluor)and incubated with platelets and PAR1-Rat1 fibroblasts. The cells werethen treated with pronase to digest extracellularly bound peptides andanalyzed by flow cytometry. Flow cytometry was conducted on platelets orRat1 fibroblasts stably transfected with PAR1 (Ishii et al., J. Biol.Chem. 269, 1125-1130 (1994)) that were treated with fluorescein-labeledpeptides, Fluor-Pal-i3 (Fluor-P1pal-19) or Fluor-i3 (Fluor-P1-i3-19) asindicated. Fluorescein was conjugated to the i3 peptides by incubatingequimolar concentrations of peptide and fluorescein-5-EX-succinimidylester (Molecular Probes) for 2 h at 25° C. in DMF/5% triethylamine. Theconjugated peptide products were purified from reactants usingreverse-phase chromatography. The composition of the conjugated peptideswas confirmed by mass spectrometry. Cells were incubated with 10micromolar Fluor-Pal-i3 or Fluor-i3 for 2 min in PBS/0.1% fetal calfserum and then treated with 2 U pronase for 15 min at 37° C. and washedprior to flow cytometry. As shown in FIG. 1E, both platelets andfibroblasts remained strongly fluorescent when treated withFluor-Pal-i3, as compared to the non-palmitoylated Fluor-i3. Otherstudies have shown that disruption of the cell membrane abrogatesprotection against pronase digestion only with Fluor-Pal-i3 and notFluor-i3, thus confirming that the palmitoylated i3 peptide is membranepermeable.

Some pepducins remain attached to the cell membrane after penetration. Afirst step to understanding the intracellular liganding of thesepepducins is to define the minimal molecular determinants of activationand the optimal position of the cell-penetrating anchor. This isaccomplished by generating compositions according to Formula I:

A-X-B-Y_(n)  (I),

wherein A is a cell-penetrating (anchor) moiety, X is a linking moiety,B is an isolated fragment of a G-protein coupled receptor (GPCR), Y is ahydrophobic moiety, such as an aromatic compound (e.g.,halogen-substituted biphenyl), a peptide or a lipid, and n is zero orone. For example, X can be a covalent bond, polyglycine, polyarginine,and a mixed sequence hydrophobic peptide. Varying the length of thelinker allows one to keep the location of the cell-penetrating (A)attachment fixed relative to activating and binding determinants in theGPCR fragment (B). In some compositions, a cell-penetrating moiety islinked to an amino acid in the GPCR fragment (B). For example, thecell-penetrating moiety can be a lipidated Cysteine.

The location of a Cys-lipid moiety (within B) is moved (by varying thelength of the linker) in the context of Pal13, with or without aN-terminal (Y) group. Dual lipidation (A and Y are both lipids) ormovement of the site of lipid attachment within B may enhance, block, ornot effect pepducin activity. Lipidation of cysteine thiols is done withN-MPB-PE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)-butyramide])by mixing 2.5 mM peptide and 5 mM N-MPB-PE (Avanti Polar Lipids) in 6%triethylamine/94% dimethylformamide and incubating at ambienttemperature for 2 h.

Example 2 Assessment of the Ability of Pepducins to Activate PAR1 in aRecombinant System

Seven GPCRs were tested (PAR1, PAR2, PAR4, CCKA, CCKB, SSTR2, and MC4)for their ability to be activated or inhibited by their cognatepepducin. Full antagonist activity was demonstrated for PAR1, PAR2 (FIG.4D), PAR4 (FIG. 4C-D), and SSTR2 ‘wild-type’ pepducins with theircognate receptors with IC50 values of 1 to 3 micromolar, as summarizedin Table 2. Of these GPCRs, PAR4 is newly-discovered (Kahn et. al.,Nature (London) 394:690-694 (1998); Xu et al., Proc. Natl. Acad. Sci.(USA) 95, 6642 (1998)). PAR4 was selected due to an interest indeveloping reagents suitable for exploring the unique ability of PAR4 tocause prolonged Ca²⁺ transients and irreversible platelet aggregation(Covic et al., (2000) Biochemistry 39, 5458). To date, the bestextracellular ligands to PAR4 bind with millimolar or high-micromolaraffinity and PAR4 inhibitors have not been reported. In FIG. 4, theanti-PAR4 pepducin, P4pal-15, inhibits PAR4 and not PAR1, whereas theconverse is true for the anti-PAR1 pepducin, P1pal-12. Thus, P4pal-15 isthe first described high-potency anti-PAR4 compound (IC₅₀=0.6 micromolarin platelets) and is currently being used to help delineate the role ofPAR4 in the vascular biology of mice. Covic et al., Nature Medicine inpress (2002).

Six of the newly tested wild-type pepducins were partial agonists fortheir own GPCR with maximal efficacies of ˜12-35% (Table 2, FIG. 7)including the P2pal-21 pepducin (FIG. 2D). However, the PAR1 pepducin,P1pal-19, robustly activates PAR2 (FIG. 2F), indicating that selectiveintroduction of mutations into P2pal-21 might create a full agonist forPAR2. An alignment of the i3 loops of PAR1 and PAR2 (FIG. 2A) revealedseveral sequence differences. A point mutation of the C-terminal lysineto phenylalanine imparted full agonist activity to the PAR2 pepducinP2pal-21F (FIG. 2D). This pepducin also activated PAR1 but not PAR4 norSSTR2 (FIG. 2G). Similar C-terminal point mutations of Lys/Arg to Pheconferred partial agonist activity to the pepducins of SSTR2, and CCKAand improved the potency of the CCKB pepducin by 15-fold (Table 2). Tosummarize, from a screen of seven diverse GPCRs, full agonists for PAR1and PAR2, partial agonists for MC4, SSTR2, CCKA, and CCKB, and fullantagonists for PAR1, PAR2, PAR4 and SSTR2 (Table 2, FIG. 7) have beendemonstrated. Thus, the pepducin inhibitors and agonists of theinvention are applicable to a broad range of GPCRs which can couple toGq, Gi, Gs, and G12/13.

The GPCR moiety of the pepducins of the present invention are derivedfrom any cells of a human being or other organism (e.g., guinea pig,rat, mouse, chicken, rabbit, pig, sheep, cattle, monkey, virus, fungi,insects, plants, bacteria, etc.), for example, splenic cell, nerve cell,glia cell, beta cell of pancreas, marrow cell, mesangial cell,Langerhans' cell, epidermic cell, epithelial cell, endothelial cell,fibroblast, fibrocyte, muscular cell, fat cell, immunocyte (e.g.,macrophage, T cell, B cell, natural killer cell, mast cell, neutrophil,basophil, eosinophilic leukocyte, monocyte, etc.), megakaryocyte,synovial cell, chondrocyte, osteocyte, osteoblast, osteoclast, mammarygland cell, hepatocyte, or interstitial cells or precursor cells, stemcells or cancer cells thereof and the like; and any tissues containingsuch cells, for example, brain, various parts of the brain (e.g.,olfactory bulb, amygdala, cerebral basal ganglia, hippocampus, thalamus,hypothalamus, substhanlamic nucleus, cerebral cortex, medulla,cerebellum, occipital pole, frontal lobe, putamen, caudate nucleus,corpus callosum, substantia nigra), spinal cord, pituitary, stomach,pancreas, kidney, liver, genital organs, thyroid gland, gallbladder,bone marrow, adrenal gland, skin, muscle, lung, digestive tract, bloodvessel, heart, thymus, spleen, submandibular gland, peripheral blood,peripheral blood leukocyte, intestinal tract, prostate, testicle,testis, ovarium, placenta, uterus, bone, joint, small intestine, largeintestine, skeletal muscle and the like, in particular, brain andvarious parts of the brain.

Cell-penetrating ability of the pepducins was evaluated as follows.Since PAR1 couples to both Gq and Gi(beta/gamma) to stimulatephospholipase C-beta (PLC-beta) (Hung et al., J. Clin. Invest. 89,1350-1353 (1992)) inositol phosphate (InsP) production in Rat1fibroblasts expressing human PAR1. Accumulation of [3H]-inositolphosphates was measured in the presence of 20 mM LiCl. Cells were splitinto 12 well plates at 200,000 cells/well. [3H]-labeled myoinositol (2μCi/mL) was added to cells 24 h prior to the experiment. Wells wererinsed twice with 2 mL DME containing 10 mM HEPES buffer, pH 7.3, thentwice with 2 mL PBS containing 20 mM LiCl. Cells were stimulated withagonist or the specified concentrations of i3-loop pepducin for 30 minand then extracted with cold methanol and chloroform. Extracts wereloaded onto columns containing 1 mL anion-exchange resin AG1X8, formateform, 100-200 mesh size (Bio-Rad Laboratories, Cambridge, Mass.). Afterloading, columns were washed twice with 10 mL H₂O and twice with 10 mL60 mM ammonium formate/5 mM Borax. Column fractions were eluted with 4mL 2 M ammonium formate/0.1 M formic acid into vials containing 7.5 mLscintillation cocktail and counted. The mean of duplicate or triplicatedeterminations was expressed as fold-stimulation above non-stimulatedcells. The biphasic pepducin data was fit to a two-site equation withone activating site (EC50) and one inhibitory site(IC50)y=(100/(1+(([peptide]/EC50)−n1)))+(100/(1+(([peptide]/IC50)−n2)))−n3by non-linear regression analysis using Kaleidagraph 3.05, where n1 andn2 are hill coefficients for the activating and inhibitory phases,respectively, and n3 is the delta maximum amplitude.

PAR1-Rat1 cells or PAR2-COS7 cells were challenged with 1 nM to 10-100μM i3 peptide or mastoparan (INLKALAALAKKIL) (SEQ ID NO:24). PLC-betaactivity was determined by measuring total [3H]-inositol phosphate(InsP) formation. As shown in FIG. 2B and C, P1pal-19, and P1pal-13which lacks the N-terminal six residues of P1pal-19, stimulate InsPproduction with EC50 values of 180±20 nM and 700±50 nM, respectively,and with similar efficacies as the natural agonist thrombin. In FIGS. 2Band 2C, PLC-β activity was converted to percent of the full responserelative to 0.1 nM thrombin (100%) and plotted as a function of peptideconcentration using a two-site equation that fit the biphasic activationand inhibition profiles. The full PAR1 thrombin responses for individualexperiments were 7.6-fold for P1pal-13, 9.4-fold for P1pal-12 andP1pal-7, 12.4-fold for P1pal-19 and P1pal-19/Rat1 alone, 18-fold forP1pal-19Q, 12.4-fold for P1pal-19E and 9.5-fold for the mastoparanexperiment. The minor stimulation of untransfected Rat1 cells (Rat1alone) by P1pal-19 in C can be attributed to the endogenous rat PAR1present in these fibroblasts since addition of SFLLRN (SEQ ID NO:23)causes similar stimulation in these untransfected cells (FIG. 2F,‘RAT1’).

Receptor stimulation of PLC-0 was determined by measuring total[³H]-inositol phospate (InsP) formation in Rat1 cells stably expressingPAR1 or in COS7 cells transiently expressing PAR2, PAR4, SSTR2, CCKA, orCCKB. Antagonist assays were conducted as in FIG. 4D: PAR1, PAR2, PAR4,or SSTR2-expressing cells were pre-treated with their cognate pepducins(10 nM-50 μM) for 5 min, and then stimulated with extracellular agonists0.1 nM thrombin, 100 μM SLIGKV (SEQ ID NO:17), 10 nM thrombin, or 1 μMAGCKNFFWKTFTSC (SEQ ID NO:18), respectively. In agonist assays, PAR1,PAR2, PAR4, SSTR2, CCKA or CCKB-expressing fibroblasts were stimulatedwith their cognate pepducins (1 nM-50 μM) for 30 min and InsP productionmeasured. The biphasic pepducin data (see FIGS. 2B-D) was fit to atwo-site equation, with an EC₅₀ for the activating phase and IC₅₀ forthe inhibitory phase. Percent efficacy was calculated relative to thefull (100%) response to extracellular agonist as above (300 nM CCK-8 forCCKA and CCKB).

TABLE 2 Agonist and Antagonist Activity of Pepducins for their Cognate Receptors Expressed in Fibroblasts. NT = not tested. SEQ AntagonistAgonist ID IC₅₀ EC₅₀ IC₅₀ Efficacy Receptor Pepducin Sequence NO (μM)(μM) (μM) (%) PAR1 P1pa1-19 Pa1-  1 —  0.18 ± 6.5 ± 90 ± 2RCLSSSAVANRSKKSRALF  0.02 1.0 P1pa1-13 Pa1-AVANRSKKSRALF  2 —  0.70 ± 32 ± 5 60-88  0.05 P1pa1-7 Pa1-KKSRALF  3 1.2 ± 0.1 — — — P1pa1-12Pa1-RCLSSSAVANRS  4 5.0 ± 1.0 — — — P1pa1- Pa1-  5 —  0.65 ±  30 ± 246 ± 8 19Q RCLSSSAVANQSQQSQALF   0.1 P1pa1- Pa1-  6 >50   2.5 ±  80 ± 511 ± 1 19E RCESSSAEANRSKKERELF   0.5 PAR2 P2pa1-21 Pa1  7 1.0 ± 0.50.018 ± 1.0 ± 18 ± 2 RMLRSSAMDENSEKKRKRAI 0.002 0.2 K P2pa1- Pa1-  8 —0.025 ±   7 ± 1 95 ± 6 21F RMLRSSAMDENSEKKRKRAI 0.003 F PAR4 P4pa110Pa1-SGRRYGHALR 38 P4pa115 Pa1-HTLAASGRRYGHALR  9 3.0 ± 1.0 — — — P4pal15Pa1-HTLAASGRRYGHALF 10  >2 — — — F SSTR2 S2pa1-23 Pa1- 11 2.0 ± 1.0 — —— KVKSSGIRVGSSKRKKSEKKV TK S2pa1- Pa1- 12 3.0 ± 1.0   0.1 ± 0.5 ± 15 ± 423F KVRSSGIRVGSSKRKKSEKKV  0.05 0.3 TF CCKA Apa1-19 Pa1- 13 NT — — —RIRSNSSAANLMAKKRVIR Apa1- Pa1- 14 NT   0.2 ±   2 ± 1 <10 19FRIRSNSSAANLMAKKRVIEF   0.1 CCKB Bpa1-18 Pa1- 15 NT   1.5 ±  10 ± 2 12 ±3 SGSRPTQAKLLAKKRVVR   0.5 Bpa1- Pa1- 16 NT  0.10 ± 1.0 ± 13 ± 2 18FSGSRPTQAKLLAKKRVVF  0.05 0.5

The activation curves of PAR1 are biphasic with a steep activating phasefollowed by a steep inhibitory phase. Splitting the P1pal-19 agonistinto C-terminal P1pal-7 and corresponding N-terminal P1pal-12 peptidesresults in loss of stimulatory activity in platelets or PAR1-Rat1 cellswhen added separately (FIGS. 1B, 1D, 2B) or together (FIG. 1B).Therefore, in order to have agonist activity, C-terminal PAR1 pepducinresidues 301-313 must be contiguous. COS7 cells were transientlytransfected with the human receptors PAR1, PAR2, PAR4, cholecystokinin A(CCKA), cholecystokinin B (CCKB), substance P (Sub-P), or ratsomatostatin receptor (SSTR2). Transfected cells were challenged with arange of concentrations (0.1-10 micromolar) of P1pal-19, P1pal-13, orP2pal-21 and the highest stimulation of the individual receptors isreported as a black column. The extracellular agonists used to definemaximum stimulation for each receptor (open column) were 10 nM thrombinfor PAR1, 100 micromolar SLIGKV (SEQ ID NO:17) for PAR2, 100 nM thrombinfor PAR4, 300 nM CCK-8 for CCKA and CCKB, 1 micromolar AGCKNFFWKTFTSC(SEQ ID NO: 18) for SSTR2, and 1.5 micromolar RPKPQQFFGLM (SEQ ID NO:25)for Sub-P. Covic et al., PNAS 99, 643 (2002).

Significantly, neither P1pal-13 nor P1pal-19 stimulate InsP(approximately 11%) in the absence of the PAR1 receptor in COS7 cells(FIG. 2E, F) or in Rat1 fibroblasts (FIG. 2C, F). These resultsdemonstrate that activation of G protein signaling by thecell-penetrating peptides requires the presence of receptor. Inaddition, positively charged residues in the C-terminal region of the i3loop peptides previously shown to be essential for activation of Gproteins (Okamoto et al., Cell 67, 723-730 (1991)) are not necessary foractivity of these membrane-tethered agonists. Substitution of thepositive charges results in only a 2-fold loss in efficacy of theP1pal-19Q peptide (FIG. 2A) in platelet aggregation (FIG. 1D) orstimulation of InsP in PAR1-Rat1 cells (FIG. 2C). Moreover, theamphipathic wasp venom peptide mastoparan, which is areceptor-independent activator of Gi/o (Higashjima et al., J. Biol.Chem. 265, 14176-14186 (1990)), did not stimulate InsP production in thePAR1-Rat1 cells (FIG. 2C). Thus, the peptides are not simply acting aspositively charged amphipathic helixes to activate G protein signalingin an uncontrolled manner. In contrast, mutation of the conserved, morehydrophobic residues in the P1pal-19E peptide (FIG. 2A) results in ˜90%loss of agonist activity (FIG. 1D, 2C).

Example 3 Specificity of Pepducins for Other GPCRs

For these PAR1-derived i3 peptides to be useful as in vivo reagents, itwas important to determine the specificity of the peptides for otherGPCRs. P1pal-19 and P1pal-13 were tested for agonist activity against anarray of six other GPCRs: PAR2, PAR4, cholecystokinin A and B (CCKA andCCKB), somatostatin (SSTR2), and substance P (Sub-P). COS7 cells weretransiently transfected with each receptor and InsP production measured.P1pal-13 is selective for PAR and did not activate the other six GPCRsincluding PAR2 (FIG. 2E). P1pal-19 can fully activate the highlyhomologous PAR2 receptor and stimulates CCKB to about 30% of its maximalactivity, but does not activate PAR4, CCKA, SSTR2, nor Sub-P (FIG. 2F).These data indicate that the P1pal-13 exhibits complementarity ofbinding to PAR1 and is highly selective. Inclusion of the six N-terminalamino acids of the i3 loop in P1pal-19 results in less selectivity.

Example 4 Construction of Agonists for GPCRs Other than PAR1

It was found in some cases that lipidated peptides, based on theircorresponding wild-type i3 sequences, were partial agonists withefficacies of 40% for MC4 (FIG. 7), 18% for PAR2 (P2pal-21, FIG. 2D) and12% for CCKB, and no agonist activity was observed for the i3 peptidesof PAR4, SSTR2 and CCKA (Table 2). However, as previously demonstrated,the P1pal-19 PAR1 peptide was able to robustly activate PAR2 (FIG. 2F)indicating that selective mutation of P2pal-21 might create a fullagonist for PAR2. An alignment of the i3 loops of PAR1 and PAR2 (FIG.2A: which shows the alignment of the third intracellular (i3) loops andadjacent transmembrane regions (TM5 and TM6) for PAR1, PAR2 and PAR4receptors with palmitoylated peptides for PAR1 and PAR2) revealedseveral sequence differences. Quite strikingly, mutation of theC-terminal Lys to Phe converts the PAR2 peptide, P2pal-21F, into apotent (EC50=25 nM), full agonist of PAR2 with biphasic properties (FIG.2D). P2pal-21F also activated PAR1 but not PAR4 nor SSTR2 (FIG. 2G).Similar C-terminal Lys/Arg to Phe point mutations of the SSTR2 and CCKApeptides conferred partial agonist activity with their cognate receptorsand improved the potency of the CCKB peptide by 15-fold.

These data suggest that the peptide must be tethered or embedded in alipophilic environment at both termini to exhibit high agonist activity.

To distinguish between indirect versus direct activation of the Gprotein by the pepducins, a point mutation was introduced at positionS309 located in the C-terminus of the i3 loop/N-terminus of TM6 of PAR1.This perimembranous region has been shown to be important for thefidelity of G protein coupling for many receptors. Cotecchia et al., J.Biol. Chem. 267, 1633-1639 (1992); Kostenis et al., Biochemistry 36,1487-1495 (1997); Kjelsberg, et al., J. Biol. Chem. 267, 1430-1433(1992). and comes into direct contact with the critical DRY residues ofTM3. Palczewski et al., Science 289, 739-45 (2000). A S309P mutant wasconstructed and transiently expressed in COS7 cells to the same level aswild type PAR1. COS7 cells were transiently transfected with wild-type(WT), S309P or delta377 PAR1 (Kuliopulos et al., Biochemistry 38,4572-4585 (1999)) receptors. Cells were challenged with P1pal-19, SFLLRN(SEQ ID NO:23), or thrombin and PLC-beta activity determined bymeasuring total [3H]-inositol phosphate formation relative to 100%stimulation (9.6-fold) of WT PAR1 with 0.1 nM thrombin. The apparentinhibition of PAR1 by very high concentrations of thrombin in B iscaused by persistent interactions of thrombin to a hirudin-like sequence(K51YEPF55) located in the e1 exodomain of PAR1 (Hung et al., J. Clin.Invest. 89:1350-1353 (1992)). High amounts of thrombin can remain boundto the thrombin-cleaved PAR1 exodomain (Jacques, et al., J. Biol. Chem.275, 40671-40678 (2000)) and inhibit intramolecular liganding by thetethered SFLLRN (SEQ ID NO:23).

The S309P mutant is deficient in thrombin- and SFLLRN-dependent (SEQ IDNO:23) stimulation of InsP with 17- and 28-fold loss of potency, and1.6- and 3.3-fold loss of efficacy, respectively (FIG. 3B, C). P1pal-19also stimulates the S309P mutant with parallel losses in potency(13-fold) and efficacy (4.3-fold) relative to its effects on wild typePAR1 (FIG. 3A). Since P1pal-19 did not correct the signaling defect ofthe S309P mutation, this indicates that the crucial C-terminal portionof the i3 region in the intact receptor exerts dominant effects incoupling to G protein over that of the exogenous pepducin.

Example 5 Determination of GPCR Regions that Interact with the Pepducins

To define the region(s) of the receptor that might directly contact thei3-pepducin, the entire C-terminal i4 domain of PAR1 was deleted(delta377). The X-ray structure of rhodopsin (Palczewski et al., Science289, 739-45 (2000)) indicates that the i3 loop may contact theN-terminal region of alpha-helix 8 and residues to the C-terminal sideof the Cys-palmitoyl moieties within the i4 C-tail. As shown in FIG. 3Band C, the delta377 mutant is defective in stimulating PLC-beta inresponse to thrombin and SFLLRN (SEQ ID NO:23). Efficacy is reduced by2-3 fold for the two PAR1 agonists and potency is shifted 22-fold forthrombin and ˜30-fold for SFLLRN (SEQ ID NO:23). In contrast, theP1pal-19 pepducin gives effectively no stimulation of PLC-beta in thepresence of the delta377 PAR1 mutant (FIG. 3A). These data demonstratethat the C-tail of PAR1 is required for P1pal-19 to activate G-proteinand that the C-tail may provide a binding surface for the pepducinagonists.

Example 6 Pepducins that Lack Agonist Activity Still Block GPCR ProteinSignaling

Human platelets were a convenient, biologically-relevant, system to testthe potency and selectivity of anti-PAR1 and anti-PAR4 pepducins sinceplatelets possess both PAR1 and PAR4 thrombin receptors with unique Ca2+signaling profiles. The PAR1 peptide, P1pal12, was found to completelyblock PAR1 signaling. Platelet Ca2+ measurements were performed as inExample 1. Platelets were pre-treated with 3 μM P1pal-12 (openarrow-head) or P4pal-15 (Pal-HTLAASGRRYGHALR (SEQ ID NO:9); closedarrow-head), and then stimulated with 3 Micromolar SFLLRN (SEQ ID NO:23)or 200 Micromolar AYPGKF (SEQ ID NO:26) as indicated. As shown in FIG.4A-C, 3 micromolar P1pal-12 effectively inhibits PAR1 activation ofhuman platelets by SFLLRN (SEQ ID NO:23), but does not block PAR4activation by AYPGKF (SEQ ID NO:26) (FIG. 4A). Moreover, a pepducincorresponding to the full-length i3 loop of PAR4, P4pal-15, had noagonist activity but was able to fully antagonize PAR4 signaling.

Platelets were then preincubated with either 3 Micromolar P1pal-12 or 3Micromolar P4pal-15 for 1 min and then challenged with 3 MicromolarSFLLRN (SEQ ID NO:23) or 200 Micromolar AYPGKF (SEQ ID NO:26) andplatelet aggregation monitored as in FIG. 1D. Full platelet aggregationtraces are also shown for the same amounts of SFLLRN (SEQ ID NO:23) orAYPGKF (SEQ ID NO:26) in the absence (−) of inhibitors. Platelets werepre-treated for 1 min with 0.01-5 Micromolar P1pal-12 or P4pal-15 andchallenged with 3 Micromolar SFLLRN (SEQ ID NO:23) or 200 MicromolarAYPGKF (SEQ ID NO:26), respectively. As shown in FIG. 4A, 3 micromolarP4pal-15 blocked AYPGKF (SEQ ID NO:26) activation of PAR4 withoutaffecting SFLLRN (SEQ ID NO:23) activation of PAR1 and is an effectiveinhibitor of platelet aggregation (FIG. 4B, C). Thus, P4pal-15 is thefirst described high-potency anti-PAR4 compound (IC50=0.6 micromolar inplatelets) and is currently being used to help delineate the role ofPAR4 in the vascular biology of mice.

PAR1, PAR4, and PAR2-expressing fibroblasts were pre-treated with0.03-100 micromolar P1pal-12, P4pal-15, or P2pal-21 for 5 min, and thenchallenged with extracellular agonists 0.1 nM thrombin, 10 nM thrombin,or 100 micromolar SLIGKV (SEQ ID NO:17), respectively. Percent InsPinhibition is calculated relative to the full extracellularagonist-stimulated response: 5.2-fold for P1pal-12, 3.1-fold forP4pal-15 and 3.1-fold for P2pal-21. Both anti-PAR1 and anti-PAR4pepducins are also able to block signaling to PLC-beta in fibroblastsexpressing PAR1 or PAR4, respectively (FIG. 4D). Lastly, the PAR2pepducin, P2pal-21, which is a partial agonist for PAR2 (FIG. 2D), isalso able to completely block PAR2 signaling in fibroblasts (FIG. 4D).

Example 7 Ligand Binding Site Peptides with C-Terminal Lipid TethersInterfere with Receptor Liganding

Peptides from the first extracellular domain (e1) PAR1 which have aC-terminal cysteine-lipid for generation of extracellular,membrane-tethered, antagonists of ligand binding to PAR1 are described.In some cases, N-terminal attachment of lipid or hydrophobic tethers tothe receptor peptide fragments may lead to loss of activity or may notbe optimally placed for targeting the receptor, G protein, or forblocking extracellular liganding. Thus, another embodiment of thistechnology is attaching lipid tethers to cysteine residues or otherderivatizable groups (i.e., —SH, —NH2, —OH) in the receptor fragmentthat are strategically located at points likely to come into membranecontact. Internal cysteines will be mutated to serine as necessary toavoid spurious derivatization. Based on molecular modeling, some of thepeptides will be lipidated at internal, N- and/or C-terminal positions.Glycine (n=1-5) or similar molecular spacers could be placed betweensites of lipidation and peptide if necessary for more efficient membraneanchoring or targeting. Dual lipidation may increase effective molarityand reduce entropic contributions at the receptor-effector orreceptor-ligand interface.

As an example, using NMR structural analysis, a region on theextracellular surface of PAR1 which forms part of the ligand bindingsite for PAR1 was identified. This region is comprised of receptorresidues P85AFIS89 and is termed ligand binding site-1 (LBS-1). Mutationof this region on PAR1 results in severe defects in receptor activationby intermolecular ligand (i.e., SFLLRN (SEQ ID NO:23)) or thrombin.Addition of lipid-tethered peptides that mimic the receptor ligandbinding site(s) might be expected to interfere with thrombin-activatedreceptor (intramolecular ligand) or exogenously added intermolecularligand (FIG. 8). Other extracellular loops of the receptor also likelymake contact with the ligand and contribute regions termed ligandbinding site-2 (LBS-2), LBS-3, etc.

A peptide based on the extracellular loop of the PAR1 receptor (LBS1:PAFISEDASGYL-C) (SEQ ID NO:27) was synthesized. This peptide containsthe P₈₅AFIS₈₉ (SEQ ID NO:31) sequence of PAR1 and adjacent C-terminalresidues E₉₀DASGYL₉₆-C(SEQ ID NO:32) that are expected to come intoclose proximity with the lipid bilayer in the intact receptor (FIG. 9B).The non-lipidated LBS1 peptide was a relatively poor antagonist againstthrombin and SFLLRN (SEQ ID NO:23) activation of PAR1-dependent plateletCa²⁺ fluxes (FIGS. 9C, and 9D, respectively). Likewise, thenon-lipidated LBS1 peptide did not inhibit 3 nM thrombin aggregation ofthe platelets (FIG. 9E). In marked contrast, the C-terminally lipidatedpeptide, LBS1-PE (FIG. 9A) was an effective inhibitor of plateletaggregation. As shown in FIG. 9E, 25 micromolar LBS1-PE completelyinhibited 3 nM thrombin-induced platelet aggregation.

The LBS1 peptide included a C-terminal cysteine residue and wassynthesized by solid-phase fmoc chemistry. The wild-type peptideE₉₀DASGYLT₉₇ (SEQ ID NO:37) was modified so that amino acid 97 (T) wasremoved and replaced with a lipidated cysteine. Lipidation of theC-terminal cysteine thiol of LBS1 was done with N-MPB-PE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)-butyramide])by mixing 2.5 mM peptide and 5 mM N-MPB-PE (Avanti Polar Lipids) in 6%triethylamine/94% dimethylformamide and incubating at ambienttemperature (23 C) for 2 h. The LBS1 peptide-Cys-PE conjugate waspurified by Sep-Pak (Waters) C18 reverse-phase chromatography, andidentity confirmed by mass spectrometry.

Example 8 Pepducin Activation of the G_(S)-Coupled MC4 Obesity Receptor

Activation of the MC4 receptor (MC4R) by melanocortin agonists, such asmelanocyte stimulating hormone (alpha-MSH) causes anorexia (loss ofappetite) and weight loss in mice. Mutations of the MC4R have been foundin extremely obese humans. A pepducin. MC4pal-14 (Pal-TGAIRQGANMKGAI)(SEQ ID NO:28) that corresponds to the third intracellular loop of thehuman MC4R was synthesized and tested the pepducin for agonist activitywith its cognate receptor. Addition of MC4pal-14 to COS7 fibroblaststransiently transfected with MC4R stimulated adenylate cyclase activityby 40% relative to authentic agonist, alpha-MSH. The activity profile ofMC4pal-14 is biphasic with an activating phase (EC50-150 nM) andinhibitory phase (IC₅₀˜10 micromolar). These data demonstrate that thepepducins can activate Gs-coupled receptor pathways and that MC4pal-14and its derivatives may have utility as anti-obesity agents in humans.Further, it is noteworthy that unlike systemically injected peptideagonists like alpha-MSH, these cell penetrating pepducins would beexpected to cross the blood-brain barrier to activate receptors such asMC4R located in the central nervous system. (FIG. 7)

Example 9 Selectivity of P1pal-12 Pepducin

P1pal-pepducin (SEQ ID NO:4) (FIG. 10A, FIG. 11F) lacked agonistactivity yet was a full antagonist of PAR1-dependent inositol phosphate(InsP) production and Ca²⁺ signaling in platelets and recombinantsystems. Covic et al., PNAS 99, 643 (2002). Preincubation of humanplatelets with 3 μM P1pal-12 for 1 min blocked 75-95% of aggregation inresponse to the PAR1 extracellular ligand SFLLRN (SEQ ID NO:23) (FIG. 11b).

The selectivity of the P1pal-12 pepducin for PAR1 versus other humanGPCRs was examined, including PAR4, thromboxane A2 (TXA₂) (Murray andFitzGerald, Proc. Natl. Acad. Sci. (USA) 86, 124 (1989)), P2Y₁ and P2Y₁₂ADP receptors (Woulfe et al., J. Clin. Invest. 107, 1503 (2001)) andnon-G-protein coupled receptors for collagen (α₂β₁, GPVI/FcγII) Moroi etal., J. Clin. Invest. 84, 1440 (1989)) and von Willebrand factor(GPIb/IX/V) (Savage et al., Cell 94, 657 (1998)) using platelets as thebiologically-relevant system. P1pal-12 was selective for PAR1 and didnot block aggregation induced by the extracellular ligands of the otherplatelet receptors. The anti-PAR1 pepducin, P1pal-12, does not inhibitPAR4-dependent platelet aggregation.

Example 10 Selectivity of P4pal-10 Pepducin

In addition to PAR1, the PAR4 receptor plays a major role in thrombinsignaling in human platelets and is responsible for formation of stableplatelet-platelet aggregates during the propagation phase ofhaemostasis. Covic et al., Biochemistry 39, 5458 (2000); and Covic etal., Thromb. Haemost. 87, 722 (2002). The palmitoylated peptide based onthe human PAR4 i3 loop, P4pal-10, Pal-SGRRYGHALR (SEQ ID NO:29) (FIG. 10a, FIG. 11 f), was synthesized.

P4pal-10 had no agonist activity as measured by platelet aggregation,intracellular Ca²⁺ release in human platelets, or InsP production inCOS7 cells transfected with hPAR4.

As shown in FIGS. 10 b-c, P1pal-12 and P4pal-10 selectively inhibitthrombin receptors on human platelets. Platelets were preincubated witheither 5 μM P1pal-12 or 5 μM P4pal-10 for 1 min and then challenged with3 μM SFLLRN (SEQ ID NO:23), 200 μM AYPGKF (SEQ ID NO:26), 20 μM U46619,5 μM ADP, 20 mg/mL collagen or 1 mg/mL ristocetin, and plateletaggregation monitored. Full platelet aggregation traces (dashed lines)are also shown for each agonist in the absence of inhibitors.

As shown in FIGS. 10D-E, human platelets were pre-treated for 1 min withthe indicated concentrations of P4pal-10 and challenged with 200 μMAYPGKF (SEQ ID NO:26) and 30 μM SFLLRN (SEQ ID NO:23) as indicated.Intracellular Ca²⁺ concentration was monitored as the ratio offluorescence excitation intensity at 340/380 nm of platelets labeledwith fura-2AM. The integrated Ca²⁺ response for the individualtreatments are relative to the full Ca²⁺ responses for each agonist(100%) recorded in the absence of P4pal-10 (top trace).

P4pal-10 was tested extensively for the ability to act as an antagonistof platelet PAR4. Preincubation of human platelets with P4pal-10completely blocked aggregation (IC₅₀=0.5-1 μM) in response to the PAR4peptide ligand, AYPGKF (SEQ ID NO:26) (FIG. 10 c). The P4pal-10 PAR4antagonist could also partially inhibit activation of PAR1 by SFLLRN(SEQ ID NO:23), but did not appreciably block aggregation in response toagonists for the TXA₂, ADP, collagen, or GPIb/IX/V receptors (FIG. 10c).

To further define the selectivity of inhibition of the P4pal-10pepducin, its ability to block PAR1 and PAR4-dependent Ca²⁺ transientsgenerated through the Gq/PLC-β signaling pathway (Offermanns et al.,Nature 389, 183 (1997)) in platelets was examined. Stimulation of PAR4with the AYPGKF (SEQ ID NO:26) agonist gives rise to a prolonged Ca²⁺signal whereas stimulation of PAR1 with SFLLRN (SEQ ID NO:23) gives adistinct spike response. Covic et al., Biochemistry 39, 5458 (2000).Preincubation of human platelets with 1.2-5 μM P4pal-10 attenuated50-80% of the PAR4 response, and 17-67% of the PAR1 Ca²⁺ signalsubsequently induced by SFLLRN (SEQ ID NO:23) (FIG. 10 d). When theorder of peptide agonists is reversed (SFLLRN (SEQ ID NO:23) followed byAYPGKF SEQ ID NO:26)) the specificity of P4pal-10 is higher in favor ofPAR4 over PAR1. As shown in FIG. 10E, 5 μM P4pal-10 inhibits only 36% ofthe PAR1 Ca²⁺ response while inhibiting 97% of the PAR4 response. ThePAR4 Ca²⁺ response to peptide agonists (FIG. 10E) is typicallydesensitized by ˜2-fold when it follows SFLLRN (SEQ ID NO:23)stimulation of PAR1. Covic et al., Biochemistry 39, 5458 (2000). Thismay explain the relative differences in magnitude of the inhibitoryeffects of P4pal-10 on the Ca²⁺ response for the two receptors dependingon the order of addition of agonists. Unlike P1pal-12, P4pal-10 caninhibit signaling from both PAR4 and PAR1 thrombin receptors with higherselectivity for PAR4 over PAR1.

Example 11 Pepducin Modulation of Platelet Aggregation

It is shown herein that P1pal-12 and P4pal-10 pepducins are effectiveinhibitors of the soluble peptide ligands of PAR1 and PAR4. Previousstudies have shown that it is considerably more difficult to generateeffective antagonists of the tethered ligands of PARs produced byproteolytic cleavage of the extracellular domains (Seiler et al., Mol.Pharm. 49, 190 (1996); and Bernatowicz et al., J. Med. Chem. 39, 4879(1996)). More recently, small molecule antagonists have been describedthat do block thrombin activation of PAR1 (Andrade-Gordon et al., J.Pharm. Exp. Therap. 298, 34 (2001); and Proc. Natl. Acad. Sci. (USA) 96,12257 (1999)) in human platelets and rodent model systems, andpeptide-ligand antagonists of PAR4 (Ma, et al., Br. J. Pharm. 134, 701(2001)) have been shown to block thrombin-induced endostatin releasefrom human platelets. Concentrations of thrombin as low as 3 nM cleavesufficient PAR1 to fully activate human platelets (Covic et al.,Biochemistry 39, 5458 (2000); and Covic et al., Thromb. Haemost. 87, 722(2002)). PAR4 is also activated by 3 nM thrombin but does not generate asufficiently strong signal to fully aggregate human platelets until thethrombin concentration exceeds ˜5 nM (Covic et al., Thromb. Haemost. 87,722 (2002)).

In FIGS. 11 a-b, aggregation was performed with human plateletspretreated for 1 min with 3 μM P1pal-12 or 3 μM P4pal-10 as indicatedand stimulated with 3 nM thrombin (T) or 20 nM thrombin. Aggregation wasmonitored as % light transmittance of stirred platelets at 37° C. Fullplatelet aggregation traces (−) are also shown for each agonist in theabsence of inhibitors. Pretreatment of human platelets with theanti-PAR1 pepducin, P1pal-12, completely blocked aggregation in responseto 3 nM thrombin (FIG. 11 a). Challenge of platelets with 20 nM thrombinwas not appreciably blocked by P1pal-12, presumably due to theadditional signal from the cleaved PAR4 receptor (FIG. 11 b).

The PAR4 pepducin, P4pal-10, also inhibited human platelet aggregationto 3 nM thrombin though not as efficiently as P1pal-12 (FIG. 11 a).Strikingly, P4pal-10 inhibited 85% of human platelet aggregation inresponse to 20 nM thrombin-conditions where normally both PAR1 and PAR4would be fully activated (Covic et al., Thromb. Haemost. 87, 722 (2002))(FIG. 11 b). The efficient blockade of thrombin-induced plateletaggregation by P4pal-10 is consistent with the inhibition data (FIG. 10c) which show that the anti-PAR4 pepducin completely blocks PAR4 andcross-inhibits PAR1. The cross-inhibition of PAR1 by the anti-PAR4pepducin may have important ramifications for the therapeutic utility ofanti-PAR pepducins by blocking a higher range of thrombinconcentrations. One possible explanation for the cross-specificity ofP4pal-10 is the sequence similarity of the C-terminal region of the i3loops in human PAR1 and PAR4, both of which include a stretch of basicresidues (FIG. 11 f). Thus, the P1pal-12 pepducin based on theN-terminal region of the PAR1 i3 loop does not appreciably cross-inhibitPAR4 which has poor homology to PAR1 in this region. Conversely, theP4pal-10 pepducin based on the more highly conserved C-terminal regionof the i3 loop has markedly higher cross-reactivity between PAR1 andPAR4.

Example 12 Pepducin Modulation of Thrombosis In Vivo

The anti-PAR4 pepducin was tested as an anti-thrombotic agent under invivo conditions using a mouse model system. The anti-PAR4 pepducin wasinvestigated rather than the anti-PAR1 pepducin because mouse plateletslack PAR1 and generate thrombin signals solely through PAR4 (Sambrano etal., Nature 413, 74 (2001)). In FIGS. 11 c-e, aggregation was performedon washed murine platelets pretreated with 3 μM P4pal-10 for 1 min andchallenged with 1-20 nM thrombin. In FIG. 11 f, alignment of the thirdintracellular loops (i3) from human and murine PAR1 and PAR4 is shown.

It was determined that the P4pal-10 pepducin based on the human PAR4 i3loop, inhibits activation of mouse platelets by thrombin. As shown inFIGS. 11C-D, 3 μM P4pal-10 completely blocks aggregation of murineplatelets by 1 nM thrombin (EC₅₀=0.6 nM) and causes a 7-fold inhibitionof the rate of aggregation to 3 nM thrombin. In addition, 3 μM P4pal-10blocked 75% of aggregation of murine platelets in response to 100 μMAYPGKF (SEQ ID NO:26), the soluble PAR4 peptide ligand. Together, thesedata suggest that the 70% identity between human and murine PAR4 in theregion of P4pal-10 (FIG. 11F) provides enough homology for the humananti-PAR4 pepducin to be effective in both species. Unlike the case inhuman platelets, however, the inhibition of murine platelets by P4pal-10is overcome at 20 nM thrombin (FIG. 11E). This may be due in part tosequence differences between human and murine PAR4 (77% overallidentity).

It was then determined that fluorescently-labeled palmitoylated i3 looppeptides based on PAR1 (fluorescently-labeled PAR4 i3 loop pepducinswere insoluble) could be delivered to circulating mouse platelets.

FIG. 12 indicates that the human P4pal-10 pepducin prolongs bleedingtime and protects against systemic platelet activation in mice. FIG. 12Ashows that the palmitoylated i3-loop peptide accumulates in circulatingmouse platelets. Palmitoylated and non-palmitoylated PAR1-i3 looppeptides were tagged with fluorescein (Covic et al., PNAS 99, 643(2002)) and injected into the mouse via a catheter in the internaljugular vein. Mice were injected with 1 μmoles/L of palmitoylated(Fluor-Pal-i3: Pal-RC(Fluor-5EX)LSSSAVANRSK(Fluor-5EX)K(Fluor-5EX)SRALF)(SEQ ID NO: 1) or non-palmitoylated (Fluor-i3:(Fluor-5EX)-NH-RSLSSSAVANRSK(Fluor-5EX)K(Fluor-5EX)SRALF) (SEQ ID NO: 1)fluorescein-labeled PAR1 i3-loop peptides. The fluorescently-labeledpeptides were allowed to circulate for 15 min, then whole blood wascollected and the platelets purified by centrifugation and treated with2 U of pronase (to remove peripherally-bound peptides) for 15 min priorto flow cytometry (Azam et al., Mol. Cell. Biol. 21, 2213 (2001), Covicet al., PNAS, 99:643-648 (2002)). 10,000 platelets were analyzed forfluorescence using a FACS Scan flow cytometer and the data plotted as ahistogram of relative cell number versus fluorescence (FIG. 12A). Flowcytometry revealed that circulating mouse platelets exposed to thepalmitoylated Fluor-Pal-i3 acquired 5-fold higher fluorescence relativeto platelets exposed to the non-palmitoylated Fluor-i3 (FIG. 12A). Thisdemonstrates that the intravenously-injected Pal-i3 loop peptides aredelivered to circulating platelets and are supportive of the proposedmechanism of FIG. 10A that palmitoylation is sufficient for delivery ofthe i3 peptide through the plasma-cell membrane. Because murineplatelets lack PAR1, yet accumulated the fluorescently-labeled PAR1 i3loop pepducin, it is unlikely that pepducins distribute to cellsaccording to the tissue-expression of their cognate receptor.

The effect of blockade of PAR4 with parenterally-administered P4pal-10on primary haemostasis was examined. It is known that knockout of thePAR4 gene results in uncontrolled tail-bleeding (Sambrano et al., Nature413: 74 (2001)). P4pal-10 (3 μmoles/L) was infused into the mousejugular vein and allowed to circulate for 5 min prior to tail-bleedmeasurements. FIG. 12B demonstrates that intravenous administration ofP4pal-10 results in rebleeding from amputated tail tips in mice. 50% ofthe mice (8/16) that were injected with P4pal-10 formed unstable thrombiat the amputated tail tips. Thus, in several cases after initialcessation of bleeding, the stream of blood reappeared after 54-280 s,and the tail re-bled for an additional 1-10 min (mean=3.2 min). There-bleeding phenomena was not observed in any of the negative controlexperiments. In these experiments, mice were infused with the anti-PARpepducin, P1pal-12 or injected with vehicle (0/17) alone. P1pal-12 (3μmoles/L, n=16), P4pal-10 (3 μmoles/L, n=16), or vehicle (DMSO, n=17)alone were allowed to circulate for 5 min prior to tail-bleedmeasurements. The re-bleeding observed in the mice treated with theanti-PAR4 pepducin is consistent with the predicted physiological roleof PAR4 in humans where PAR4 has been shown to control the stability ofplatelet-platelet aggregates (Covic et al., Biochemistry 39, 5458(2000); and Covic et al., Thromb. Haemost. 87, 722 (2002)).

The total mean bleeding time was markedly prolonged in the mice treatedwith P4pal-10 as compared to mice injected with vehicle alone (FIG.12C). FIG. 12C shows that blocking PAR4 with P4pal-10 extendstail-bleeding time in mice, the mean total bleeding times±2 S.E. areshown as open circles (P<0.002). The mean total bleeding time of theP4pal-10-treated mice was 249 s, compared to 119 s for thevehicle-treated mice or 95 s for the P1pal-12-treated mice.

Thus, treatment of mice with P1pal-12 (3 μmoles/L) did not prolongtail-bleeding time or result in unstable haemostasis (FIGS. 12B-C) aswas observed with P4pal-10. The PAR1 pepducin antagonist was predictedto have no effect on haemostasis since platelets that are derived frommice genetically deficient in PAR1 have no defects in thrombin signalingand aggregation and have normal bleeding times (Connolly et al., Nature381: 516 (1996)).

In FIG. 12 D, platelets were systemically activated by intravenousinfusion of an AYPGKF (SEQ ID NO:26)/epinephrine cocktail without orwith varying amounts of P4pal-10, and platelet count was measured. Asshown in the figure, the PAR4 pepducin P4pal-10 protects againstsystemic platelet activation in mice. P4pal-10 (0.3-3 μmoles/L) orvehicle alone (DMSO) were delivered intravenously and allowed tocirculate for 5 min.

Example 13 Pepducins as Protective Agents In Vivo

The efficacy of the anti-PAR4 pepducin as a protective agent againstsystemic platelet activation in vivo was assessed. Intravascularplatelet activation was induced with a cocktail containing the PAR4agonist AYPGKF (SEQ ID NO:23) plus epinephrine. Epinephrine activatesthe G_(i(z))-coupled α₂-adrenergic receptor (Woulfe et al., J. Clin.Invest. 107, 1503 (2001)) which serves to potentiate the response ofG_(q)-coupled receptors like PAR4. Infusion of the platelet agonistcocktail caused a precipitous drop in mean platelet count from 900×10³to 300×10³/μL (FIG. 12 d) which is due to incorporation of plateletsinto systemic thrombi (Fabre et al., Nat. Med. 5:1199 (1999); and Smythet al., Blood 15:1055 (2001)). In contrast, pre-infusion with 0.3μmoles/L of P4pal-10 provided 36% protection against systemic thrombusformation (P<0.08). Higher concentrations of P4pal-10 (1 and 3 μmoles/L)resulted in 45-70% protection against systemic platelet activation(P<0.01-0.003). The protective effect of blockade of PAR4 with theP4pal-10 pepducin strongly correlates with the results obtained withmice genetically-deficient in PAR4 which were shown to be resistant toarteriolar thrombosis (Sambrano et al., Nature 413:74 (2001)). Together,these data demonstrate that pharmacologic blockade of PAR4 with theP4pal-10 pepducin protects against systemic platelet activation andsuggest that PAR4 may be an important target in the prevention ofthrombosis in humans.

The deployment of i3-loop pepducins provides a simple and powerfulapproach to determine the effect of pharmacologic disruption of GPCRswhich lack a known extracellular antagonist. The application ofpepducins in in vivo model systems, such as mice, is useful to validateand extend the information generated from genetic knockout of a GPCR,particularly in cases where embryonic lethality resulting from geneticdisruption of the GPCR precludes analysis in the mature animal.

Example 14 Inhibition of the P2Y₁₂ ADP Nucleotide Receptor withPepducins

Recently, the P2Y₁₂ adenosine diphosphate (ADP) receptor was cloned bythree independent groups (Hollopeter et al., Nature 409:202 (2001);Zhang et al., J. Biol. Chem. 276:8605 (2001); and Takasaki et al., Mol.Pharmacol. 60:432 (2001)) and shown to be the long-sought after targetof the thienopyridine drugs clopidogrel and ticlopidine (Gachet et al.,Biochem Pharmacol 40:2683 (1990); Mills et al., Arterioscler Thromb12:430 (1992)). These anti-P2Y₁₂ drugs are highly effectiveantithrombotic agents for the treatment of patients with acute coronarysyndromes. P2Y₁₂ plays such a critical role in thrombus formationbecause it can modulate the strength of platelet-platelet adhesiveinteractions via multiple receptors such as PAR1. Inputs from P2Y₁₂stabilize the critical transition point of platelet-platelet aggregatesduring the progression from early reversible GPIIb-IIIa-fibrinogenbinding to the late phase of irreversible binding to fibrinogen. Forexample, the PAR1 agonist, SFLLRN (SEQ ID NO: 23), causes full andirreversible platelet aggregation (FIG. 13A). If the ADP nucleosidaseapyrase is added, aggregation becomes reversible to SFLLRN (SEQ ID NO:23). This is because apyrase destroys the ADP that is released from theplatelet dense granules and the P2Y₁₂ ADP receptor can no longerstabilize the platelet-platelet aggregates initiated by addition of thePAR1 agonist, SFLLRN (SEQ ID NO: 23). Pepducins based on the i3-loop ofP2Y₁₂ (Y12pal-18 and Y12pal-24) were made, and were determined to haveno agonist activity in platelet aggregation assays (FIG. 13A).

Y12pal-24 was an effective inhibitor of irreversible plateletaggregation (FIG. 13B) and at 10 μM was equivalent in efficacy asaddition of saturating amounts of apyrase which completely ablates theADP autocrine response (FIG. 13A). Human platelets (gel-purified) wereactivated with 3 μM SFLLRN (SEQ ID NO: 23) in the presence of 0, 1, 3,or 10 μM pepducin. Aggregometry was performed by light scattering.

These data demonstrate that pepducins can be made to inhibitnucleotide-liganded GPCRs which further underscores their generalapplicability across the various classes of GPCRs.

Example 15 Activation and Inhibition of a Class B GPCR (Glucagon-LikePeptide Receptor-1) with Pepducins

Glucose-induced insulin secretion is modulated by a number of hormonesand neurotransmitters. In particular, two gut hormones, glucagon-likepeptide-1 (GLP-1) and gastric inhibitory peptide (GIP) potentiate theeffect of glucose on insulin secretion and are thus calledgluco-incretins (Dupre, The Endocrine Pancreas, E. Samois Ed. (RavenPress, New York, 253-281 (1991)) and Ebert and Creutzfeld, DiabetesMetab. Rev. 3, (1987)). Glucagon-like peptide-1 is a gluco-incretin bothin rat and in man (Kreymann et al., Lancet 2:1300 (1987)). It is part ofthe preproglucagon molecule (Bell et al., Nature 304:368 (1983)) whichis proteolytically processed in intestinal L cells to GLP-1(1-37) andGLP-1 (7-36)amide or GLP-1(7-37) (Mojsov et al., J. Biol. Chem.261:11880 (1986) and Habener et al., The Endocrine Pancreas E. SamoisEd. Raven Press, New York 53-71 (1991)). The stimulatory effect of thesegluco-incretin hormones is mediated by activation of adenylate cyclaseand a rise in the intracellular concentration of cyclic AMP (Drucker etal., Proc. Natl. Acad. Sci. USA 84:3434 (1987) and G'ke et al., Am. J.Physiol. 257:G397 (1989)). GLP-1 has also a stimulatory effect oninsulin gene transcription (Drucker et al., Proc. Natl. Acad. Sci. USA84: 3434 (1987)). In a rat model, non-insulin-dependent diabetesmellitus (NIDDM) is associated with a reduced stimulatory effect ofGLP-1 on glucose-induced insulin secretion (Suzuki et al., Diabetes39:1320 (1990)). In humans, in one study, GLP-1 levels were elevated inNIDDM patients both in the basal state and after glucose ingestion;however, following a glucose load there was only a very small rise inplasma insulin concentration (Qrskov et al., J. Clin. Invest. 87:415(1991)). Another recent study (Nathan et al., Diabetes Care 15: 270(1992)) showed that GLP-1 infusion could ameliorate postprandial insulinsecretion and glucose disposal in NIDDM patients.

Pepducins based on the i3 loop of glucagon-like peptide receptor-1(GLP-1R) were generated, and both activated and inhibited this class BGPCR. As shown in FIG. 14 A, 1 μM G1pal-15 pepducin (SEQ ID NO:35)caused 1.7-fold activation of GLP-1R as measured by enhancement of cAMPin Cos7 fibroblasts transiently transfected with human GLP-1R (cAMPaccumulation was measured by radioimmunoassay in a 96 well plate).Conversely, at higher concentration (10 μM, FIG. 14 B), both G1pal-23(SEQ ID NO:36) and G1pal-15 (SEQ ID NO:35) inhibited 40-60% of theactivity of GLP-1R in response to its authentic ligand, 1 nM GLP-1peptide.

Example 16 Inhibition of Chemoinvasion of Metastatic Breast Cancer Cellsby PAR1 and PAR2-Based Pepducins

Recently, PAR1 has been shown to act as a chemokine receptor ininflammatory cells and its expression is tightly correlated withmetastatic propensity of breast cancer cells. Experiments were performedin order to determine if application of pepducins based on the i3 loopsof PAR1, PAR2, or PAR4 would inhibit migration and invasion of breastcancer cells. It was determined that the highly invasive MDA-MB-231breast cancer cell line expressed very high levels of functional PAR1,PAR2 and PAR4. As shown in FIG. 15, addition of the PAR1 pepducinantagonist, P1pal-7 (SEQ ID NO:3), inhibited chemoinvasion of MDA-MB-231cells by 30%. Notably, addition of the PAR2 i3 loop pepducin P2pal-21F(SEQ ID NO:8) inhibited 90% of chemoinvasion of the breast cancer cells.In contrast, the anti-PAR4 pepducin, P4pal-10 (SEQ ID NO:29) stimulatedchemoinvasion by 40%, suggesting that P4pal-10 has chemokine activitypotentially via PAR4. These findings suggest that pepducins targetedtowards GPCRs that are expressed in cancer cells could prove beneficialin halting the progression of invasive cancer. Pepducins were added withNIH3T3 Conditioned medium (CM) to the bottom well of a Transwellapparatus.

Example 17 Inhibition of Trypsin Activation of the PAR2 Receptor with anAnti-PAR2 Pepducin

PAR2 is rapidly attracting interest from researchers in the field ofchronic inflammation. The cellular responses to PAR2 activation aregenerally pro-inflammatory. Whenever tissues in the body are disturbedan inflammatory response is mounted in order to protect the tissues frompotential pathogen entry so that normal healing process can proceed.When inappropriately activated, chronic inflammatory diseases result.For example, in asthma, inappropriate activation occurs in response to afalse (non-infectious) airway stimulus, while in chronic obstructivepulmonary disease (COPD), the inappropriate stimulus is cigarette smoke,industrial chemicals, or notably—a genetic defect in α1-antitrypsin.PAR2 is selectively activated by trypsin and trypsin-like enzymes andimportantly, PAR2 is expressed ubiquitously by barrier cells of avariety of organs, i.e. epithelia and endothelia. The epithelialexpression of PAR2 is particularly striking and leads to the suggestionthat PAR2 may be involved in defensive reactions at these barriers.Cells that express PAR2 in the human lung include epithelial cells,airway smooth muscle and fibroblasts, as well as vascular smooth muscleand endothelial cells. PAR2 is also known to be expressed by human mastcells, macrophages and neutrophils, kerotinocytes and myocytes.Moreover, PAR2 is expressed in the epithelia of the small and largeintestines, the pancreatic duct, and myenteric neurons where it mayparticipate in a variety of intestinal inflammatory syndromes and invisceral pain. PAR2 is coexpressed with pro-inflammatory neuropeptidessubstance P (SP) and calcitonin gene-related peptide (CGRP) on sensorynerves, where it mediates neurogenic inflammation. New evidence nowlinks peripheral PAR2 activation to hyperalgesic responses via releaseof SP in the spinal cord. In summary, therapeutic exploitation ofanti-PAR2 compounds might have relevance in the generation of newanalgesics, anti-inflammatories, anti-asthmatics and anti-proliferativeagents.

The ability of the anti-PAR2 pepducin, p2pal-21, was tested for itsability to antagonize one of the authentic protease agonists of PAR2,namely trypsin. As shown in FIG. 16, 3 μM p2pal-21 is able to shift theEC50 of trypsin activation of PAR2 (as measured by InsP production inCos7 stably expressing PAR2) by 10-fold. Thus, the p2pal-21 pepducin caninhibit both the intramolecularly-activated PAR2 (FIG. 16) and theintermolecularly-activated PAR2 (FIG. 4D).

Example 18 Screening and Detection Methods

The composition of the invention can be used to screen drugs orcompounds that modulate GPCR activity or expression as well as to treatdisorders characterized by insufficient or excessive production of GPCRprotein or production of GPCR protein forms that have decreased oraberrant activity compared to GPCR wild-type protein.

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)that bind to GPCRs or have a stimulatory or inhibitory effect on, e.g.,GPCR protein expression or GPCR activity. The invention also includescompounds identified in the screening assays described herein.

The invention provides assays for screening candidate or test compoundswhich bind to or modulate the activity of the membrane-bound form of apepducin-GPCR complex or biologically-active portion thereof. The testcompounds of the invention can be obtained using any of the numerousapproaches in combinatorial library methods known in the art, includingfor example, biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds. See, e.g., Lam, 1997. Anticancer DrugDesign 12: 145.

A “small molecule” as used herein, is meant to refer to a compositionthat has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be, e.g., nucleic acids,peptides, polypeptides, peptidomimetics, carbohydrates, lipids or otherorganic or inorganic molecules. Libraries of chemical and/or biologicalmixtures, such as fungal, bacterial, or algal extracts, are known in theart and can be screened with any of the assays of the invention.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt, et al., 1993. Proc. Natl.Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci.U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho,et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem.Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed.Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (e.g., Houghten,1992. Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354:82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner, U.S. Pat. No. 5,233,409),plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89: 1865-1869)or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990.Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci.U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310; Ladner.U.S. Pat. No. 5,233,409).

An assay is a cell-based assay in which a cell which expresses amembrane-bound form of a GPCR, or a biologically-active portion thereofon the cell surface, plus a pepducin, is contacted with a test compoundand the ability of the test compound to bind to the GPCR and displacethe pepducin determined. The test compound could bind at theextracellular surface, transmembrane domains, or intracellular surfacesof the GPCR target and inhibit or enhance the pepducin activation of theGPCR. The cell, for example, is of mammalian origin or a yeast cell.Determining the ability of the test compound to displace the pepducinfrom the GPCR protein can be accomplished, for example, by coupling thepepducin to a radioisotope or enzymatic label such that binding of thetest compound displaces the pepducin from the GPCR orbiologically-active portion thereof. Alternatively, the test compoundscan be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly orindirectly, and the pepducin could displace the radio-labeled testcompound from the GPCR and the free radio-labeled test compound detectedby direct counting of radioemission or by scintillation counting.Alternatively, test compounds can be enzymatically-labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by increases or decreases in conversionof an appropriate substrate to product upon addition of pepducin.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a membrane-bound form of GPCR protein, or abiologically-active portion thereof, on the cell surface with a testcompound and determining the ability of the test compound to modulate(e.g., stimulate or inhibit) the binding, activity of the pepducin forthe GPCR As used herein, a “target molecule” is a molecule with which aGPCR protein binds or interacts in nature, for example, a molecule onthe surface of a cell which expresses a GPCR interacting protein, amolecule on the surface of a second cell, a molecule in theextracellular milieu, a molecule associated with the internal surface ofa cell membrane or a cytoplasmic molecule. A GPCR target molecule can bea non-GPCR molecule or a GPCR peptide of the invention. In oneembodiment, a GPCR target molecule is a component of a signaltransduction pathway that facilitates transduction of an extracellularsignal (e.g. a signal generated by binding of a compound to amembrane-bound GPCR) through the cell membrane and into the cell. Thetarget, for example, can be a second intercellular protein that hascatalytic activity or a protein that facilitates the association ofdownstream signaling molecules with GPCR.

Determining the ability of the test molecule to interact with a GPCRtarget molecule can be accomplished by one of the methods describedabove for determining direct binding. In one embodiment, determining theability of the test molecule to inhibit the GPCR peptide interactionwith a GPCR target molecule can be accomplished by determining theactivity of the target GCPR-pepducin complex. For example, the activityof the target molecule can be determined by inhibiting GPCR-peptideinduction of a cellular second messenger of the GPCR target (i.e.intracellular Ca²⁺, diacylglycerol, IP₃, etc.), detectingcatalytic/enzymatic activity dependent on GPCR activation or inhibition,detecting the induction or inhibition of a reporter gene (comprising aGPCR-responsive regulatory element operatively linked to a nucleic acidencoding a detectable marker, e.g., luciferase), or detecting a cellularresponse, for example, cell survival, cellular differentiation, or cellproliferation.

Alternatively, an assay of the invention is a cell-free assay comprisingcontacting a GPCR peptide or biologically-active portion thereof with atest compound and determining the ability of the test compound to bindor modulate (e.g. stimulate or inhibit) the activity of the GPCR proteinor biologically-active portion thereof.

Binding of the test compound to the GPCR can be determined eitherdirectly or indirectly as described above. For example, the assaycomprises contacting the pepducin plus the GPCR or biologically-activeportion thereof with a known compound which binds GPCR to form an assaymixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to interact with a GPCRprotein, wherein determining the ability of the test compound tointeract with a GPCR protein comprises determining the ability of thetest compound to preferentially bind to GPCR or biologically-activeportion thereof as compared to the known compound.

Determining the ability of the test compound to modulate the activity ofGPCR can be accomplished, for example, by determining the ability of theGPCR peptide to bind to a GPCR target molecule by one of the methodsdescribed above for determining direct binding. Alternatively,determining the ability of the test compound to modulate the activity ofGPCR peptide can be accomplished by determining the ability of the GPCRpeptide to further modulate a GPCR target molecule. For example, thecatalytic/enzymatic activity of the target molecule on an appropriatesubstrate can be determined as described above.

The cell-free assay comprises contacting the GPCR peptide orbiologically-active portion thereof with a known compound which bindsthe GPCR to form an assay mixture, contacting the assay mixture with atest compound, and determining the ability of the test compound tointeract with a GPCR, wherein determining the ability of the testcompound to interact with a GPCR comprises determining the ability ofthe GPCR peptide to preferentially bind to or modulate the activity of aGPCR target molecule.

The cell-free assays of the invention are amenable to use of both thesoluble form or the membrane-bound form of GPCR protein. In the case ofcell-free assays comprising the membrane-bound form of GPCR protein, itmay be desirable to utilize a solubilizing agent such that themembrane-bound form of GPCR protein is maintained in solution. Examplesof such solubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate,3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS), or3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate(CHAPSO).

It may be desirable to immobilize either GPCR peptide or its targetmolecule to facilitate separation of complexed from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of a test compound to GPCR protein, or interaction ofGPCR protein with a pepducin in the presence and absence of a candidatecompound, can be accomplished in any vessel suitable for containing thereactants. Examples of such vessels include microtiter plates, testtubes, and micro-centrifuge tubes. In one embodiment, a fusion proteincan be provided that adds a domain that allows one or both of theproteins to be bound to a matrix. For example, GST-GPCR fusion peptidesor GST-target fusion proteins can be adsorbed onto glutathione sepharosebeads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatizedmicrotiter plates, that are then combined with the test compound or thetest compound and either the non-adsorbed target protein or GPCRpeptide, and the mixture is incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads or microtiter plate wells are washed toremove any unbound components, the matrix immobilized in the case ofbeads, complex determined either directly or indirectly, for example, asdescribed, vide supra. Alternatively, the complexes can be dissociatedfrom the matrix, and the level of GPCR peptide binding or activitydetermined using standard techniques.

Other techniques for immobilizing proteins on matrices are also used inthe screening assays of the invention. For example, either the GPCRpeptide or its target molecule can be immobilized utilizing conjugationof biotin and streptavidin. Biotinylated GPCR peptide or targetmolecules can be prepared from biotin-NHS (N-hydroxy-succinimide) usingtechniques well-known within the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with GPCR peptide or target molecules, but which donot interfere with binding of the GPCR peptide to its cognate GPCR, canbe derivatized to the wells of the plate, and unbound target or GPCRpeptide trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the GPCR peptide or target molecule, as well asenzyme-linked assays that rely on detecting an enzymatic activityassociated with the GPCR peptide or target molecule.

Modulators of GPCR protein expression are identified in a method whereina cell is contacted with a candidate compound and the expression of GPCRmRNA or protein in the cell is determined. The level of expression ofGPCR mRNA or protein in the presence of the candidate compound iscompared to the level of expression of GPCR mRNA or protein in theabsence of the candidate compound. The candidate compound can then beidentified as a modulator of GPCR mRNA or protein expression based uponthis comparison. For example, when expression of GPCR mRNA or protein isgreater (i.e., statistically significantly greater) in the presence ofthe candidate compound than in its absence, the candidate compound isidentified as a stimulator of GPCR mRNA or protein expression.Alternatively, when expression of GPCR mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of GPCR mRNA or protein expression. The level of GPCR mRNA orprotein expression in the cells can be determined by methods describedherein for detecting GPCR mRNA or protein.

The peptide sequences discussed herein are presented in Table 3.

TABLE 3 SEQUENCES SEQ ID NO: SEQUENCE Name/Identifier  1RCLSSSAVANRSKKSRALF P1Pa119  2 AVANRSKKSRALF P1Pa113  3 KKSRALF P1Pa17 4 RCLSSSAVANRS P1Pa112  5 RCLSSSAVANQSQQSQALF P1Pa119Q  6RCESSSAEANRSKKERELF P1Pa119E  7 RMLRSSAMDENSEKKRRRAIK P2Pa121  8RMLRSSAMDENSLKKRKRAIF P2Pa121F  9 HTLAASGRRYGHALR P4Pa115 10HTLAASGRRYGHALF P4Pal15F 11 KVKSSGIRVGSSKRKKSEKKVTK S2Pa123 12KVRSSGIRVGSSKRKKSEKKVTF S2Pa123F 13 RIRSNSSAANLMAKKRVIR APal19 14RIRSNSSAANLMAKKRVIEF APa119F 15 SGSRPTQAKLLAKKRVVR BPa118 16SGSRPTQAKLLAKKRVVF BPa118F 17 SLIGKV PAR2 Extracellular Agonist 18AGCKNFFWKTFTSC Somatostatin Receptor Extracellular Agonist 19RELTLGLRFDSDSDSQSRVRNQGGLPGAVHQNGRCRPETGAVGE CCKB i3 loopDSDGCTVQLPRSRPALELTALTAPGPGSGSRPTQAKLLAKKRVV R 20LELYQGIKFEASQKKSAKERKPSTTSSGNYEDSDGCYLKTRPPR CCKA i3 loopKLELRQLSTGSSSRANRIRSNSSAANLMAKKRVIR 21 ITLWASEIPGDSSDRYHEQVSAKRKVVKSubP i3 loop 22 KVKSSGIRVGSSKRKKSEKKVTR SSTR2 i3 loop 23 SFLLRNPAR1 Extracellular Ligand 24 INLKALAALAKKIL Mastoparan (waspvenom peptide) 25 RPKPQQFFGLM SubP agonist 26 AYPGKF PAR4 Agonist 27PAFISEDASGYLC LBS-1 28 TGAIRQGANMKGAI MC4pa1-14 29 SGRRYGHALR P4Pa110 30RALAANGQRYSHALR murine PAR4 i3 loop 31 PAFIS fragment for LBS-1 32EDASGYLC modified fragment for LBS-1 33 YVRTRGVGKVPRKKVNVF Y12pa1-18 34KELYRS YVRTRGVGKVPRKKVNVF Y12pa1-24 35 ANLMSKTDIKCRLAF G1pa1-15 36SIVVSKLKANLMSKTDIKCRLAF G1pa1-23 37 EDASGYLT WT fragment for LBS-1 38MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTI CXCR4YSIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS 39 IFLPTIYSIIFLTGIVGNGLVILVTM1 of CXCR4 40 YCYVSII P1-i3-26 41 RLAF Transmembrane domain of P1pa17

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1-22. (canceled)
 23. A method for identifying an agent that modulatesthe activity or function of a G protein coupled receptor (GPCR), themethod comprising: a. providing a cell comprising said GPCR, or having aproperty or function ascribable to said GPCR; b. contacting said cellwith a polypeptide, said polypeptide comprising: i. a first domaincomprising an intracellular loop, or a fragment thereof, of said GPCR,wherein said fragment comprises at least 3 contiguous amino acidresidues of the intracellular loop; and ii. a second domain, attached tothe first domain, wherein said second domain comprises acell-penetrating, membrane-tethering hydrophobic moiety comprising: alipid, cholesterol, a phospholipid, a steroid, a sphingosine, aceramide, octyl-glycine, 2-cyclohexylalanine, or benzolylphenylalanine;wherein said polypeptide is an agonist or antagonist of said GPCR; andc. contacting said cell with a candidate agent; d. determining whethersaid candidate agent alters the activity or function of said GPCR;wherein, an alternation in the activity or function of said GPCR in thepresence of said candidate agent, a as compared to a control in which acell is not exposed said candidate agent, is indicative that said agentmodulates the activity or function of said GPCR.
 24. The method of claim23, wherein said polypeptide is an agonist of its cognate GPCR.
 25. Themethod of claim 24, wherein a decrease in the activity or function ofsaid GPCR in the presence of said candidate agent, a as compared to acontrol in which a cell is not exposed said candidate agent, isindicative that said agent is an antagonist of said GPCR.
 26. The methodof claim 23, wherein said activity or function of the GPCR is determinedby a second messenger assay.
 27. The method of claim 26, wherein saidsecond messenger is intracellular Ca²⁺, diacylglycerol, or IP₃.
 28. Themethod of claim 23, wherein said GPCR is a Class A or Class B GPCR. 29.The method of claim 23, wherein said GPCR is a mammalian GPCR.
 30. Themethod of claim 23, wherein the second domain comprises a moiety that isa tridecanoyl (C13), myristoyl (C14), pentadecanoyl (C15), palmitoyl(C16), phytanoyl (methyl substituted C16), heptadecanoyl (C17), stearoyl(C18), nonadecanoyl (C19), arachidoyl (C20), heniecosanoyl (C21),behenoyl (C22), trucisanoyl (C23), or lignoceroyl (C24) moiety.
 31. Themethod of claim 23, wherein the second domain comprises a moiety that isa palmitoyl (C16), pentadecanoyl (C15) or myristoyl (C14) moiety.
 32. Amethod for identifying an agent that modulates the activity or functionof a G protein coupled receptor (GPCR), the method comprising: a.providing a composition comprising said GPCR in membrane-bound form; b.contacting said membrane-bound GPCR with a polypeptide, said polypeptidecomprising: i. a first domain comprising an intracellular loop, or afragment thereof, of said GPCR, wherein said fragment comprises at least3 contiguous amino acid residues of the intracellular loop; and ii. asecond domain, attached to the first domain, wherein said second domaincomprises a membrane-tethering hydrophobic moiety comprising: a lipid,cholesterol, a phospholipid, a steroid, a sphingosine, a ceramide,octyl-glycine, 2-cyclohexylalanine, or benzolylphenylalanine; whereinsaid polypeptide is an agonist or antagonist of said GPCR; and c.contacting said membrane-bound GPCR with a candidate agent; d.determining whether said candidate agent alters the activity or functionof said GPCR; wherein, an alternation in the activity or function ofsaid GPCR in the presence of said candidate agent, a as compared to acontrol in which said GPCR is not exposed said candidate agent, isindicative that said agent modulates the activity or function of saidGPCR.
 33. The method of claim 32, wherein said polypeptide is an agonistof its cognate GPCR.
 34. The method of claim 33, wherein a decrease inthe activity or function of said GPCR in the presence of said candidateagent, a as compared to a control in which a cell is not exposed saidcandidate agent, is indicative that said agent is an antagonist of saidGPCR.
 35. The method of claim 32, wherein said activity or function ofthe GPCR is determined by a second messenger assay.
 36. The method ofclaim 35, wherein said second messenger is intracellular Ca²⁺,diacylglycerol, or IP₃.
 37. The method of claim 32, wherein said GPCR isa Class A or Class B GPCR.
 38. The method of claim 32, wherein said GPCRis a mammalian GPCR.
 39. The method of claim 32, wherein the seconddomain comprises a moiety that is a tridecanoyl (C13), myristoyl (C14),pentadecanoyl (C15), palmitoyl (C16), phytanoyl (methyl substitutedC16), heptadecanoyl (C17), stearoyl (C18), nonadecanoyl (C19),arachidoyl (C20), heniecosanoyl (C21), behenoyl (C22), trucisanoyl(C23), or lignoceroyl (C24) moiety.
 40. The method of claim 32, whereinthe second domain comprises a moiety that is a palmitoyl (C16),pentadecanoyl (C15) or myristoyl (C14) moiety.